Welcome to the OLI version of Modern Biology. This introductory course is called "Modern Biology" because it is focused on topics at the forefront of experimentation in the fields of cellular biology, molecular biology, biochemistry, and genetics. It is carefully planned to provide the background students will need for advanced biology classes. Students from other disciplines may also find this course useful as it explains many of the concepts and techniques currently discussed in the popular press and applied in other contexts.

Fluorescent image of a living mouse cell
This is a mouse tissue culture cell showing the cytoskeletal structure. The large clearing in the center is the nucleus. Image courtesy of Jon Jarvik, Department of Biological Sciences, Carnegie Mellon University.

An image of a living mouse cell in culture is shown above. As the cell adheres to the bottom of the culture dish it spreads out exploring the local environment giving it the angular shape. The cell can move, utilize energy, and divide to produce new cells. During this course you will explore the fundamentals of how a cell is able to carry out each of these processes. Modern biology is about the molecular events that occur inside a cell: the making of proteins, the building of cellular structures, and the interaction of a cell with its environment.

This Modern Biology course is built around several key concepts that provide unifying explanations for how and why structures are formed and processes occur in a biological system. Because it is not possible to cover the breadth of modern molecular biology in one semester, an understanding of these key concepts will provide a basis for extension of your knowledge to biological systems beyond the specific topics covered in this course. One of the major goals of the course therefore is for you to not only learn the fundamentals of the concepts but also to recognize how they can be applied in other contexts. Several key concepts include:

The course is organized into units covering the areas of basic biochemistry, cell biology, and molecular and cellular function. The first unit introduces the basic chemistry of a cell. All other units will rely heavily on the concepts and background introduced in this unit. You are encouraged to master this material before proceeding to the other units.

Before you begin you may want to read the General Instructions about the course.

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To put the course in perspective, we begin by exploring the cell and the components of the cell called organelles. The focus of this course is to understand the components of the cell, how they interact with each other, how they are created, destroyed and how they regulate transport, growth and division of the cell. We will examine the controlled chemical environment a cell maintains and what restrictions this places on the types of chemical reactions it can perform. This background is vital to understanding key processes such as how a cell releases energy from glucose, makes and folds proteins, and goes through growth and cell division.

Above is a caricature of a eukaryotic cell. Many of the cell components are hyper links that will provide you with an image showing these same structures in a living cell. This illustration highlights one of the goals of the course which is to expand your view of biology by bridging from classic simple illustrations to images generated from actual data. In addition, you will develop an understanding of the fundamental processes used in this imaging method.

Practice

This exercise uses the Cell to let you test your knowledge of the functions of the organelles.

Biological systems use only a small subset of the elements (approximately 10 %) found in the periodic table. The chemical reactions that take place in cells represent only a small subset of all possible reactions. Before we begin an in-depth study of other aspects of molecular and cellular biology, one needs to understand the restrictions the cellular environment places on the possible chemical reactions and the resulting structures. By learning about the characteristics of the subset atoms, and a limited set of functional groups found in biological molecules, you will be able to identify, and predict many of the reactions that can take place, understand and predict the physical properties of the molecules made by these reactions, and develop an understanding of why a process occurs as it does in a cell.

Roll over each axis for details on the constraints biological systems make on that axis. The blue cube represents the chemical universe as defined by temperature (in Kelvin) on one axis, pH on another, and the elements on the third. The small orange interior cube represents the part of chemistry that occurs in cells. 310K is average human body temperature (37˚C or 98.6˚F).

We start with an understanding that the cellular environment is essentially aqueous (water) based, and thus, we will begin with a discussion of the chemistry of water as it relates to bonding, pH and temperature control. The first two concepts (bonding and pH) dictate why molecules are soluble in the cell, in what part of the cell they are soluble, what charge molecules will carry and how that charge is controlled by pH.

In the next few pages, we discuss each of the 10 learning objectives described in the objective link (check mark icon at the top and bottom of each page). Each subsequent page will list the specific objectives relevant to that page. Many of the simulations and demonstrations, used in this module, are referenced in the Glossary Module.

Atoms of Life

The key biologically relevant elements are hydrogen (H), carbon (C), nitrogen (N), oxygen (O), phosphorous (P), and sulfur (S). These elements represent over 95% of the mass of a cell. Carbon is a major component of nearly all biological molecules. Some elements are found in relatively small amounts and are called “trace elements.” The examples include sodium (Na), potassium (K), chlorine (Cl), manganese (Mn), Zinc (Zn). Throughout the course you will see how atoms of these elements are very important to the functioning of a cell. Living organisms get the required elements from outside and constantly rearrange these elements to build their own molecules. Thus, understanding behavior and structure of elements is important for understanding life.

Elements are characterized by their atomic structure. While the subatomic structure of the atom is a major topic of interest in chemistry, physics and biophysics, the basic structure described below provides sufficient information for the construction of molecules in the context of this course. Atoms are made up of subatomic particles: protons, neutrons, and electrons. Protons and neutrons are at the center of the atom and have a mass of 1 atomic mass unit (a.m.u) each. Each proton has a positive charge (+1), while neutrons are neutral (they carry no charge). Each electron has a negative charge (-1) and zero mass. Two atoms that differ by the number of neutrons are called isotopes of the same element (e.g. radioactive isotope of iodine is used for cancer treatment).

These elements represent over 95% of the mass of a cell. Carbon is a major component of nearly all biological molecules. Elements are characterized by their atomic structure. While the subatomic structure of the atom is a major topic of interest in chemistry, physics and biophysics, the basic structure described below provides sufficient information for the construction of molecules in the context of this course. Atomic mass, the sum of the number of protons and neutrons in the atomic structure, is a particularly useful measure of each element. By summing the atomic mass of all the atoms in a molecule, one can estimate the molecular mass of the molecule, which is then expressed in atomic mass units or Daltons. The masses of the six atoms of the elements listed above are given in the following Atomic Properties TABLE. The masses can be found in the upper right hand corner of the box for each element in the periodic table.

Atomic Properties of the Major Biological Atoms
Atom Mass
H 1
C 12
N 14
O 16
P 31
S 32
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Atoms Form Molecules

One characteristic of the atoms of the major elements is that they are able to form molecules through formation of covalent bonds with other atoms.

Covalent bonds
(Definition)
Covalent bonds represent the sharing of the electrons (negatively charged subatomic particles between atoms.) The number of covalent bonds that can form is dictated by the number of unpaired electrons in the outer valence shell of the atom.

Each atom in a molecule will complete its outer shell of electrons, which is 2 for hydrogen, and 8 for second row elements (e.g. C, N and O). The valence shells for each of the biologically relevant elements are highlighted in the periodic table below. The relationship between the number of unpaired electrons in the valence shell and the number of possible covalent bonds an element can form is given in the Atomic Properties TABLE above.

Only the valence shells are shown. The six shaded elements have unpaired electrons and readily form covalent bonds.

Molecules are made up of atoms covalently bound to each other. For example, a molecule of methane is a carbon atom covalently bonded to four hydrogen atoms; and water is composed of an oxygen atom covalently bonded to two hydrogen atoms. Molecules can also be complex containing many atoms covalently bonded to each other, for example, cholesterol. Note that cholesterol also contains bonds that involve the sharing of more than one valence electron between two atoms creating a double bond indicated by C=C. In this case, each carbon still only participates in making four bonds. This will be discussed later in the section on bonding.

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Electronegativity

Another property of the atoms is electronegativity.

Electronegativity
(Definition)
The tendency of an atom to attract electrons to itself. Electronegativity increases as one moves from left to right, across the periodic chart. Because the electronegative atoms have the potential to attract electrons, i.e., the electrons spend more time on the electronegative atom, a molecule containing an electronegative atom will have partial negative charge associated with that atom (as indicated by δ-). The the bonding partner in the covalent bond becomes partially positively charged (as indicated by δ+ ).
The arrow shows the tendency of atoms of the various elements to lose, keep, or gain an electron.

As you can see in the table, oxygen, nitrogen, and sulfur are electronegative atoms and represent the major electronegative atoms in biological molecules. Phosphorous, an important component in nucleic acids (e.g. DNA) is also electronegative. Water is a good example of how the very electronegative atom of oxygen influences the electrons shared with hydrogen. Oxygen has six valence electrons; two sets of paired electrons and two sets of single bonding electrons. The shared electron pair of each oxygen-hydrogen covalent bond, illustrated below, spends more time associated with the oxygen atom than the hydrogen atom. This results in the oxygen being partially negative, and the hydrogen partially positive, as indicated in the figure below by δ- and δ+. These are then described as a polar covalent bonds. The presence of polar covalent bonds in water and in other molecules containing electronegative atoms puts these molecules in a family of molecules referred to as being polar. We will return to polar bonds in water and their consequences in the "Importance of Water" section. Oxygen is not the only electronegative element that forms polar bonds, as long as the electronegativities of the two atoms differ, the bond will have some degree of polar character. For example, the P-O bond in DNA is polar, with the phosphorous having a partial positive charge since it is less electronegative than oxygen.

Because oxygen is strongly electronegative, it draws the electrons, e-, it shares with the hydrogen atoms, to itself creating a charge imbalance as indicated by the bold arrow. The oxygen is slightly negative and the hydrogen is slightly positive. This imparts a polar characteristic to the oxygen-hydrogen bond.

The Ionic State

Ionic Bond
(Definition)
An ion is an atom or a molecule that carries a charge. Negatively charged ions, called anions, form when a neutral molecule or atom gains one or more electrons. Positively charged ions, called cations, form when a neutral molecule or atom looses one or more electrons. Ionic bonds are interactions between oppositely charged ions. An ionic bond forms due to an attraction between a positive and a negative ion. No electron sharing occurs in the ionic bond.

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There are trace amounts of many other elements in cells most of which exist as ions. These include sodium (Na+), chlorine (Cl-), fluorine (F-), iron (Fe++), magnesium (Mg++), cobalt (Co++), and manganese (Mn++). Throughout the course you will see how atoms of these elements are very important to the functioning of a cell.

As you can see in the periodic table these atoms are found on the extreme right or the extreme left of the periodic table. For example, chlorine is on the right of the periodic table and is extremely electronegative and, thus, wants to acquire an electron, whereas, sodium on the extreme left of the periodic table wants to give up its unpaired electron. By losing an electron, sodium becomes a positively charged cation and by gaining an electron, chlorine becomes a negatively charged anion. These charged atoms are called ions and form the basis for charge repulsion and attraction in the non-covalent ionic bond. The affinity of Na+ for Cl- is a non-covalent, ionic bond (the attraction of opposite charges).

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Water and Hydrogen Bonding

Water is the solvent of life on Earth. It has several properties that contribute to its suitability to support life as we know it. One property derived from the special properties of oxygen is that water is a polar molecule. Oxygen is electronegative and draws the electrons that it shares in the covalent bond with hydrogen towards itself.

The electronegative oxygen (red) draws electrons to it, creating the partial negative charge on oxygen and partial positive charge on hydrogen.

Water Molecular Structures

In pure water, the partially negative oxygen of one molecule attracts the partially positive hydrogens from another water molecules to form a non-covalent bonding interaction called a hydrogen bond.

Hydrogen Bonding
(Definition)
The attraction of an electronegative atom for a hydrogen that is covalently bonded to another electronegative atom. This involves the attraction of a hydrogen with a partial positive charge to an atom with a partial negative charge. However, only hydrogens covalently bonded to an electronegative atom can participate in hydrogen bonding.
Adjust the volume on your computer and click the play button.
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Answering questions like the one above is good practice for answering the kinds of questions that may appear on the exams. Your answers may also shape discussions in class.

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Molecular Bonding

Three major types of chemical bonding have been described thus far: covalent bonding, and two forms of non-covalent bonding, ionic and hydrogen. These bonds are all important in the functioning of a cell.

Covalent Bonds

Covalent bonds are the strongest. One atom fully shares one, two or three electrons with another atom., forming a single, double, or triple bond, respectively. The bonds can be between the same element (e.g., C-C bonds) or between different elements (e.g., C-O, C-N, H-O). The nature of the covalent bond is determined by the number of electrons shared and the nature of the two elements attached.

Single bonds: Two atoms attached by a single covalent bond have free rotation about the bond.

Double bonds: Two carbons attached by a double covalent bond can only undergo 180 degree rotations and the atoms bound to these carbons are constrained to lie in the same plane as the carbon atom.. Rotation about the carbon-carbon bond has structural implications for molecules in which they are found. For example, the type of bond can influence the fluidity of biological membranes and restrict the folding of proteins.

Triple bonds. Although carbon can form triple bonds, such as in the compound acetylene (HCCH), triple bonds are not found in biological systems.

Aromatic Compounds: Involve the sharing of electrons between atoms that form a ring. The shared electrons form a partial (1/2) double bond between each atom in the ring structure. Aromatic compounds have unique geometrical properties and absorb ultraviolet light .

Formation of a covalent bond
A covalent bond is formed between two hydrogen atoms.

Covalent bonds involving electronegative atoms often result in polar molecules. As discussed earlier, the bonds between the hydrogen and oxygen atoms of water are polar, due to the fact that oxygen is electron withdrawing and hydrogen is willing to give up its lone electron. The double bond between carbon and oxygen atoms in a carbonyl group is polar, for the same reason that the oxygen attracts the electrons to itself.

Geometry

The overall shape of molecules depends on the geometry of the bonds that are formed between atoms. The shape of a molecule can have a large effect on its biological activity, often small changes in the shape of a molecule will make it biologically inactive.

We saw with water that the orientation of atoms around the oxygen was tetrahydral with the angle between the two hydrogen atoms close what would be expected for a tetrahydral shape. Both carbon and nitrogen also form tetrahydral shapes. In the case of methane the four hydrogen atoms are at the corners of a tetrahydron. In ammonia, the three attached hydrogens form the base of a tetrahydron. In the ammonium ion, the addition of a fourth hydrogen, to form NH4+, places the fourth hydrogen at the last unoccupied corner of the tetrahydron.

Carbon and nitrogen can also form planer geometries, where all of the atoms that are bound to the carbon (or nitrogen) are in the same plane as the carbon or nitrogen. In the case of carbon, a planer geometry is usually observed if the carbon is involved in a double bond, while the tetrahydral geometry is found if the carbon is forming single bonds. Compare the structure of methanol to formaldhyde in the Jmol below. In methanol, carbon forms four single bonds while in formaldehyde there is a double bond between the carbon and the oxygen. The carbon atom in methanol shows tetrahdyral geometry, while the carbon atom in formaldehdye is planer; the two hydrogens and oxygen lie in the same plane as the carbon..

Chirality

An important aspect of carbon bonding is the fact that carbon can covalently bond to four groups and that the bonding geometry of the carbon atom is tetrahedral. Therefore, if the four groups attached to the carbon are different, then two unique arrangements of the groups around the carbon atom are possible and this carbon is said to be an asymmetric center or a chiral center. The two arrangements of groups about the carbon are mirror images of each other and these two structures are referred to as enantiomers. It is impossible to superimpose these two mirror images, consequently they are distinct molecules.

Because the enantiomers have identical functional groups attached to the chiral center they have identical physical and chemical properties - except for the direction they rotate plane polarized light. Experimentally, the enantiomers are distinguished by the direction of rotation of plane polarized light. The enantiomer that rotates light to the right is designated as D (dextro) form of the compound. The other enantiomer will rotate light in the opposite direction, and is designated as the L (levulo) form of the compound.

The example shown below is the three carbon carbohydrate, glyceraldehyde. This compound contains a chiral center at the middle, or second carbon, because that carbon has four different groups attached to it. The configuration of atoms in the left-hand structure causes polarized light to be rotated to the right and is therefore the D form. Its mirror image shown on the right, rotates polarized light in the opposite direction, and is therefore the L form. Note that in both compounds the H and OH groups project out of the page towards you, but in the D form the -OH group is to the right of carbon two while in the L-form it points to the left.

The two arrangements of atoms around a chiral center are mirror images much as the right hand is a mirror image of the left. The dark black arrows indicate that atoms are above the page and the gray arrows indicate the atoms are below the page.

While enantiomers usually undergo chemical reactions in an identical fashion, biological systems are capable of discriminating between these structurally different molecules because biological systems themselves contain chiral centers, such as in amino acids. The ability to discriminate between the enantiomers is an example of bioselectivity. The chemical world has been narrowed by the selective use of specific enantiomers (in this case only D-glyceraldehyde) by biological systems.

chirality
(Definition)
When identical groups attached to a carbon are arranged in multiple ways such that two of the resulting structures are non-superimposable, they are mirror images of each other.

Non-Covalent Bonds

Ionic Bonds

Ionic bonds form between oppositely charged atoms. No electron sharing or transfer occurs. The atoms are attracted to each other due to their opposite charges. For example, the positive Na ion, and negatively charged Cl ion, are attracted to each other and form table salt. In an aqueous solution, these ions are completely dissociated and are defined as strong electrolytes. In water molecules surround the ions to form polar interactions to satisfy the charges on the ions. Thus the ions become encapsulated by water spheres, which are called spheres of hydration. The biological world is very ionic and the spheres of hydration are important in a cell because they maintain the separation of the many ions of the cell from each other. The sphere of hydration must be broken in order for binding to take place with a specific binding partner.

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Previously, water was described as having a high dielectric constant. This property that is a measure of the polarity of the covalent bond within the molecule accounts for the separation of ions by polar molecules such as water. The force of attraction between two oppositely charged ions is inversely proportional to the dielectric constant. Thus water with a high dielectric constant decreases the attraction between opposite charges. This is physically explained by the ability of polar solvents to form ordered hydration layers around ions.

Hydrogen Bonding

Hydrogen bonding was covered in Water -- Hydrogen Bonding (go there now.) Remember that hydrogen bonding occurs between partially negatively charged electronegative atoms, and partially positively charged hydrogen atoms that are attached to electronegative atoms such as oxygen, nitrogen or sulfur. Hydrogen bonding is a critical bonding in the cell. It is the principal bonding that holds the tertiary structure of proteins, carbohydrates and nucleic acids together and the overall stability of these molecules is due in part to the cumulative effect of the large number of hydrogen bonds found in the functional structures. Hydrogen bonds are found in and between a variety of molecules. For example, the enormous number of hydrogen bonds between strands of cellulose provide the strength and structure of the plant cell wall.

Hydrophilic Interaction

The nature of polar molecules is that they contain electronegative atoms, consequently they are capable of hydrogen bonding with aqueous or polar solvents. Because polar molecules are generally water soluble, they are referred to as being hydrophilic, or water-loving. The one-carbon alcohol, methanol, is an example of a polar molecule.

Hydrophobic Interaction

The final type of interaction occurs between neutral, hydrophobic, or water-fearing, molecules. These molecules do not interact with water and are characterized by a complete lack of electronegative atoms. In aqueous solutions the hydrophobic molecules are driven together to the exclusion of water. For example, shaking a bottle of oil and vinegar (acetic acid in water), such as in a salad dressing, results in the oil being dispersed as tiny droplets in the vinegar. As the mixture settles, the oil collects in larger and larger drops until it only exists as a layer, or phase, above the vinegar.

A similar effect occurs in biological systems. As a protein folds to its final three-dimensional structure, the hydrophobic parts of the protein are forced together and away from the aqueous environment of the cell. Similarly, biological membranes are stabilized by the exclusion of water between layers of lipids as we will see later.

The hydrophobic effect does not involve direct bonding between the non-polar molecules, it is an entropy driven process. You may recall that processes that increase the disorder of a system are more favorable. When a hydrophobic molecule is truly dissolved in water, the water forms a highly ordered ice-like shell around the compound. When the hydrophobic molecules contact each during separation of the aqueous and non-polar phases, the ordered water is released and become highly disordered. The increase in disorder of the released water molecules is responsible for the spontaneous assembly of many biological systems, such as proteins and membranes.

Mixed Non-polar/polar molecules: Of course, there are instances where even molecules with electronegative atoms will not be water soluble. Computer algorithms are currently used to predict water solubility based on structure. For our purposes, we will balance the ratio of polar and non-polar elements in a structure to estimate the chemical nature of any compound we are going to study.

Amphipathic molecules are molecules that have a distinct non-polar, or hydrophobic region, and a distinct polar region. These molecules do not form true solution is water. Rather, the non-polar parts are forced together into a non-polar aggregate, leaving the polar part of the molecule to interact with the aqueous phase. Detergents and long-chain carboxylic acids are examples of amphipathic molecules.

van der Waals Interactions

An important force in biochemistry is due to van der Waals interactions. This interaction occurs between any two surfaces that are in contact. The force is actually an electrostatic one that occurs as a result of a momentary fluctuation in the charge on one surface.. This charge causes the other surface to momentarily assume the opposite charge, leading to a net attractive force. If one of the surfaces has a permanent dipole, due to the presence of electronegative or electropositive atoms, then the attraction is stronger. The strength of van der Waals forces depend on the contact surface area; the larger the area the larger the interaction. At the molecular level, van der Waals interactions can contribute 10s of kJ/mol of energy. At the macroscopic level van der Waals forces can become quite large. For example, the common gecko generates sufficient van der Waals forces due to the large surface area of its foot pads to walk on the ceiling!

Origin of van der Waals effects. Two neutral surfaces (left) have no net attraction. One surface becomes charged for a short period of time. The charge on one surface generates a charge of the opposite sign on the other surface, leading to an attractive force between the two surfaces. A short time later the charges reverse in sign, again generating an attractive force between the two surfaces.

Energy Associated with the Bonds

Each of the bond types represents a measurable amount of energy. To break a bond, the equivalent amount of energy must be expended. In metabolism, bonds are broken in molecules, such as glucose, to "release" the energy. The cell utilizes this energy to drive other energy consuming reactions. The covalent bond has the most energy associated with it, on average approximately 100 kilocalories/mole (kcal/mol). The non-covalent bonds, ionic and hydrogen, and hydrophobic interactions, have approximately 5 kcals/mol associated with each of them.

It should be noted here that throughout the presentation of this course approximations will be used for certain values so that estimations can be made as we move to more complex systems. It is to be acknowledged that very precise values for each of the measurements are not available.

Thus the non-covalent bonds that have been introduced have approximately 20 times less energy associated with them and, thus, are more easily broken individually. However, hydrogen bonds generally form extensive networks, and the total energy associated with the network is the sum of the individual interactions. As anyone who has done a "belly buster" knows, breaking a large surface area of water is extremely difficult (and painful!).

Energy Associated with the Different Bonds
Bond Energy, kcal/mol
Covalent 100
Ionic 5
Hydrogen 5
Hydrophobic interactions 5
van der Waals 5 (depends on surface area)

When NaCl dissolves in water, each atom becomes surrounded by at least 20 water molecules. As NaCl there is 5 kcal/mol of energy associated with the ionic attraction of the cation and anion, but when a Na ion is surrounded by 20 water molecules, there is 100 kcal/mol of energy associated with just the Na ion. Thus, NaCl in an aqueous solution is energetically more favored than NaCl as the ionically bonded molecule due to the resulting hydrated state. You will explore what happens to molecules that only partially dissociate, or weak electrolytes, in water in the next module.

Practice

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Biological systems use only a small part of the total chemical repertoire. This is due to the number of chemical reactions that can occur under physiological conditions, and to the small number of chemical or functional groups compatible in biological systems. The physical properties of these groups define how molecules behave in biological systems at physiological pH and temperature. By learning the properties of these groups, you will be able to predict how various molecules will function, as well as which functional groups can be converted to others during metabolism

The functional groups fall into three broad categories: non-polar, polar neutral, and polar charged. The molecules in each group all have common properties.

Functional Group Tutorial

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If you need a reminder of chemical structures or how to view molecules in 3-D, use the link below.

Practice

This exercise uses the Functional Group Glossary to let you test your knowledge of the properties of the functional groups.

Check Your Understanding

In the previous module you learned about a limited set of functional groups on molecules found in biological systems and some of their properties. Specifically you learned there were only three types of functional groups: non-polar, polar neutral, and polar charged. Within these types there are eight that are particularly relevant to biological systems out of the hundreds of known organic functional groups. Of the three types, the polar charged groups, amino, carboxyl, and phosphate, undergo the greatest change depending on the environment in which they are found. They are weak electrolytes and behave as bases (amino groups) or acids (carboxyl and phosphate groups) and as such have the potential to exist as ions. In this module you will explore the properties of weak acids and bases including their dissociation equilibrium and their ability to act as pH buffers. The cell exploits these properties to control intracellular pH, the ionic state of molecules and the activity level of molecules. Thus we need to have a thorough understanding of the chemical behavior of acids and bases in solution. In the final module of this unit you will see these concepts in action in protein ligand binding.

pH

The hydrogen ion concentration, [H+] of a solution is an important property because biological systems contains functional groups whose properties are changed by changes in the hydrogen ion concentration.

Since the hydrogen ion concentations are usually much less than one, and can vary over many orders of magnitude, a different scale is used for the hydrogen ion concentration, the pH scale. The pH is the -log of the proton concentration:

pH = - log [H+].
The log conversion reduces a 10 fold change in hydrogen ion concentration to a one unit change in pH. The minus sign changes the negative numbers that would be obtained from log[H+] to positive ones. Since the pH scale is an inverse scale the concentration of protons is high at low pH and low at high pH. A solution is said to be acidic if the pH is less than seven, and basic if the pH is above 7. A solution is neutral if its pH is equal to 7.0.

The image below shows the pH of a number of common fluids.

pH of various compounds.
On the left are biological compounds and on the right are some foods and cleaning products.

Differences in hydrogen concentration

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Acid Dissociation and pH

For our studies, the Bronsted definition of an acid will be used, we will define an acid as a proton donor and a base as a proton acceptor. Hydrochloric acid, like sodium chloride, is a strong electrolyte because it completely dissociates in aqueous solution into charged ions. Hydrochloric acid is also a strong acid because when it completely dissociates it also completely donates all of its protons.

Many molecules are weak electrolytes and exist in an equilibrium (indicated by in the general equation below) between the starting molecule and its dissociated parts. Thus dissociation can be seen as an acid (HA) in equilibrium with a proton (H+) and the corresponding conjugate base (A-).

In general:

HA A- + H+

Specifically for Acetic acid:

CH3COOH CH3COO- + H+

Practice Quiz

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Acid Dissociation and Equilibrium

In this, and following, sections, we will begin to develop a quantitative relationship between the strength of an acid and how the acids ionization state will depend on the pH of the solution. When placed in water, acetic acid dissociates into the conjugate base, acetate, and a proton.

CH3COOH CH3COO- + H+
The extent to which the acid will dissociate in pure water is expressed as Ka, the equilibrium constant for dissociation of an acid:
Ka=[CH3COO][H+][CH3COOH]

Ka is the ratio of the mathematical product of the concentration of each product of the reaction (in this case the charged species) to the concentration of the reactants (in this case the neutral species). The square brackets [ ] around the terms indicate concentration and is usually expressed in molar concentration (moles/Liter). In the Learn By Doing you will explore the equilibrium of an acid dissociation in pure water. In this simulation you can assume the concentration is molecules per beaker and thus the concentration will be equal to the total number of molecules in the experiment.

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Ka is constant for an acid.

In the tutorial you learned that the equilibrium constant Ka is the same for an acid regardless of the starting concentration. Ka describes the relationship between the concentration of the protonated acid and the charged dissociated species at equilibrium. You also saw that even though Ka is constant, the values of the concentration of each species was varying over time. After an initial rapid dissociation of the acid, an equilibrium was reached. At equilibrium, the number of individual molecules will fluctuate, but on average, a balance, or equilibrium mixture, is maintained. The balance of products and reactants is defined by Ka.

Each acid has its own Ka

The equilibrium constant, Ka, is a property of the molecule and its environment. The chemical nature of the molecule has the largest effect on Ka. Secondary to this, the environment of the group can shift the Ka. Therefore, every acidic group has its own Ka value. For the purposes of this course, we will use the approximation that Ka is the same for all molecules with the same functional group. The table below lists the Ka values for several acids.

Ka for various acids
Acid Ka
Acetic Acid 0.0001
Boric Acid 0.000000001
Ammonia (NH4+) 0.000000001

Relationship of Ka and pKa

The Ka is a small number, usually much less than 1, and can vary by several orders of magnitude from compound to compound. For example, acetic acid has a Ka of around 0.0001 or 1.0 x 10-4 while the Ka of ammonia is 105 fold smaller. Due to the wide range of Ka, it is more practical to represent the Ka on a minus log scale, in the same way as the hydrogen ion concentration. Consequently, the negative log of the Ka is used to represent acidity constants:

pKa = -log(Ka)

A change in the pKa from 5 to a pKa 4 is the change by a factor of 10 in the Ka. The table below will help you see the relationship between Ka and pKa. The pKa is a convenient scale for comparing the dissociation constants of weak acids because the pKa scale is similar to the pH scale. The relationship between the two will be explained in the next few pages. Remember that the pKa is a property of the acidic group while pH is a property of the solution.

Ka pKa
0.1 1
0.01 2
0.001 3
0.0001 4
0.00001 5

Just as the pH scale indicates the relative proton concentration of various solutions, the pKa indicates the relative strengths of the different acids. If a reaction has a large equilibrium constant, then the concentration of the products will be higher than the reactants. In the case of the acidity constant, a larger Ka indicates a more completely dissociated acid, or a stronger acid. Since the pKa = - log (Ka), strong acids will have small pKa values.

For this course you do not need to memorize the pKa values for all of the different groups. The values will always be given to you, or you can find them in the Functional Groups Glossary. However, it is useful to remember the following pKa values:

Acidic Group pKa Example
-COOH (carboxyl) 4.0 Aspartic acid side chain.
-NH3 (amino) 9.0 Lysine side chain

Control of Acid Dissociation

Placing acetic acid in pure water establishes an equilibrium between the charged and neutral forms according to the unique dissociation constant of the acid. This occurs because acetic acid is the only proton donor in the solution. What would happen if protons are added to the solution from another source, for example a strong acid, or a biochemical reaction that releases protons? Answer the question before continuing.

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Because the equilibrium constant Ka does not change, the presence of additional protons from another source must shift the ratio of acetate ions to acetic acid, and a new equilibrium point would be reached.

For the following acid dissociation:

HA A- + H+
with an equilibrium constant of:
Ka=[A][H+][HA]
How will the equilibrium position change when the proton concentration changes? If protons are added [A-] must decrease and [HA] increase in order for Ka to remain the same. Likewise if protons are removed by adding a strong base then [A-] must increase, and [HA] decrease to keep Ka constant. The ratio of [A-] to [HA] changes depending on the total [H+] of the solution.

To quantify how the equilibrium point is affected by pH, it is instructive to rearrange the equilibrium equation to generate the Hendersson-Hasselbach equation. First we start with the general equilibrium equation for Ka:

K=[A-][H+][HA]
and then take the -log of both sides of the equation.
log(K)= log([A-][HA])log([H+])
You may recognize that -log(Ka) is pKa and the -log([H+]) is pH. By substituting these into the equation we have the Hendersson-Hasselbach equation.
pH=pKa+ log([A-][HA])
This equation can be used to calculate the ratio of [A-]/[HA] for any weak acid, provided the pKa of the acid and the pH of the solution are known.

Effect of pH on Protonation State

The fraction of a group that is protonated: [HA]/{[HA]+[A-]} can be calculated at any pH using the Hendersson-Hasselbach equation. Such a plot is shown below for acetic acid. Note that:

  • When pH = pKa, one half of the molecules will be protonated
  • When the pH is one unit lower than the pKa 90% of the molecules will be protonated.
  • When the pH is one unit higher than the pKa only 10% of the molecules are protonated.
  • For pH values outside +/-1 pH unit from the pKa the group is essentially fully protonated (pH << pKa) or fully deprotonated (pH >> pKa)
The fraction protonated as a function of pH is plotted for a weak acid with a pKa=4.0, such as acetic acid. Note that when the ph=pKaone-half of the molecules will be protonated. More than half are protonated with the pH is lower than the pKa while less than half are protonated when the pH is greater than the pKa.

Buffers

Weak acids can act as pH buffers when the pH is within approximately one unit of the pKa for the acid. A buffer solution will resist changes in pH as a strong acid or base is added to the solution. The following shows the pH of a solution of a weak acid as a strong base, e.g. hydroxide, is added or titrated into the solution.

The pH of a solution of a weak acid with a pKa of 4.0 is shown as base is added to the solution. The initial pH of the solution is low, and most of the weak acid is fully protonated. As base is added the weak acid remains protonated, consequently the added base causes a rapid rise in pH due to the neutralization of protons. When the pH of the solution is near the pKa of the weak acid, the acid begins to dissociate, producing protons that neutralize the added base. Since the base is being neutralized the pH climbs more slowly. When the pH is above the pKa most to the weak acid is deprotonated and can no longer provide protons to neutralize the base, consequently the pH climbs rapidly again. The pH region that is within one unit of the pKa is considered to be the buffer region.

Understanding the reverse titration, the addition of a strong acid to a solution of conjugate base follows the same logic. At high pH, the added protons do not protonate the weak acid since the pH is much higher than the pKa. In the buffer region of pH = pKa+/-1, the protons that are added to the solution will not decrease the pH, instead they will convert some of the conjugate base, A-, to the acid form, HA. At the lower edge of the buffer region the weak acid is almost fully protonated (pH=pKa-1), consequently it cannot absorb any additional protons.

The buffering of pH plays an important role in the normal function of the cell. If the pH drops too low, or becomes too high, the cell can no longer function. The principal buffer that is used in biological systems is the carbonic acid, a weak acid with an effective pKa of 6.4, ideally suited to act as a buffer when the pH ~ 7.0

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So far, we have discussed the major elements and functional groups that are important in the functioning of a cell. Together these elements and functional groups define the major properties of the four classes of macromolecules that make up a cell: carbohydrates, proteins, lipids and nucleic acids. In this module, we will explore two of these classes: carbohydrates and proteins.

Carbohydrates, proteins and nucleic acids are all examples of polymers. These polymers are very large molecules composed of smaller units joined by covalent bonds using a common set of chemical reactions. Proteins are linear polymers of amino acids all joined by peptide bonds. Polysaccharides are the carbohydrates joined through glycosidic bonds in sometimes quite complex branched structures. Later in the course you will encounter DNA and RNA which are polymers of nucleic acids linked by phosphodiester bonds. This unit includes a discussion of the structures of polymers of carbohydrates and of amino acids.

Carbohydrates

One of the simplest of the biological molecules is the carbohydrate. The name is descriptive of the character of this class of molecules since they all have the general formula of a hydrated carbon.

(C(H2O))n

We are starting with this class of molecules because they are the basis for the classical naming of the enantiomers encountered in biological molecules. But before we examine the structure of these compounds, let us look at some of the basic uses/functions of the carbohydrates.

The primary function of carbohydrates is as a source of energy. You will recall that molecules are a collection of atoms connected by covalent bonds. In general, single covalent bonds can be represented as having approximately 100 Kcal/mole of energy associated with the force that holds the two atoms together. The most common carbohydrate in nature is glucose, which has the general formula (C(H2O))6 or C6H12O6. When glucose is completely metabolized in a cell, 673 kcal of energy is released for each mole of glucose. The net equation for this process of glucose oxidation can be written as follows:

(C(H2O))6 + 6 O2 -------------> 6 CO2 + 6 H2O + 673 kcal (energy)

The challenge for the cell is to capture released energy and convert it to a useful form so that it can do work (e.g. power movement or build new macromolecules). How cells manage to do this is discussed in the Metabolism section of the course.

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The second function for the carbohydrates is structure. In this case, structure is not only what a polymer of the carbohydrates has, but it is also what that structure contributes to the cell. For example, cellulose is a linear polymer of glucose that interacts with other cellulose polymers to form fibers that interact to form the basic structure of the cell wall of plants.

Another example is the peptido-glycan that makes up the cell wall structure of a bacterium. Peptido refers to a peptide, which is a fragment of a protein or a short polymer of amino acids, and glycan refers to a polysaccharide, a polymer of carbohydrates. In this case, the polymer of carbohydrates includes building blocks other than glucose, but the end result is the formation of a matrix. In both cases the resulting fibers and matrices provide scaffolding that gives rigidity (structure) and protection to the organisms with which they are associated.

A third function for carbohydrates is that of cell recognition and signaling. Just as we identified a peptido-glycan as a conjunction (conjugation) of a peptide with a polysaccharide, other complex carbohydrates are conjugated to other molecules to form glycoproteins (carbohydrates linked to proteins) and glycolipids (carbohydrates linked to lipids). Because a very large number of structures can be made from a few monosaccharides (simple carbohydrates), a very large number of different structures can be made from a few simple carbohydrates, as will be seen later. This large number of different structures can therefore be used as unique signals for identification of individual cell types.

Video Lecture

Monosaccharides

The simplest of the carbohydrates fall into two categories or structures that differ only in the arrangement of the atoms as seen below. In fact, in biological systems it is quite easy to convert between the two forms using catalysts. The unique functional group associated with that category defines each of the categories.

In the case of the structure on the left, the number 1 carbon (the top carbon) contains the carbonyl that is flanked by a hydrogen and a carbon thus making this an aldehyde. This category of carbohydrates is, thus, referred to as aldoses. In contrast, the structure on the right has its carbonyl at the number 2 carbon (the center carbon) and this carbonyl is flanked by carbons on both sides, thus, making this carbonyl a ketone. Carbohydrates containing this ketone group are referred to as ketoses.

Aldoses
(Definition)
A category of simple carbohydrates where the number 1 carbon (the top carbon) contains the carbonyl that is flanked by a hydrogen and a carbon thus making this an aldehyde.
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Glyceraldehyde was chosen as the first carbohydrate to study because it provides another example of the bioselectivity of biological systems. In this case the selectivity is based on structural discrimination and will apply to all of the carbohydrates.

Examine the glyceraldehyde structure given above and determine which of the carbons has a unique composition of four bonding partners. The number 1 carbon has three bonding partners: an oxygen that is double bonded to the carbon, a hydrogen, and the remainder of the structure. The number 2 carbon has an aldehyde, a hydrogen, a hydroxyl group and the rest of the molecule attached, while the number three carbon has a hydroxyl, two hydrogens and the upper portion of the molecule attached. The number 2 carbon isthe only carbon that has its four bonds involved with four different groups and, thus, is a unique carbon identified as a chiral or asymmetric center. Based on the fact that this chiral carbon has tetrahedral bonding structure, it can form structural enantiomers.

You should convince yourself that it is not possible to superimpose the bottom two structures. They are in fact different structures. For glyceraldehyde, where A is an aldehyde, B is a hydrogen and X is the hydroxymethyl (CH2OH) group, the structure on the left would be referred to as D-glyceraldehyde and the structure on the right would be referred to as L-glyceraldehyde based on the orientation of the hydroxyl functional group.

Biological systems are selective and while it is possible to synthesize both of the glyceraldehyde enantiomers in the chemistry lab, a cell primarily produces and uses the D form of glyceraldehyde. This is dictated by the way in which molecules are selected and used in a cell. Every reaction in a cell is catalyzed by an enzyme and these enzyme catalysts have the ability to discriminate between different structures. In this case the enzymes can discriminate between D and L carbohydrates. Thus as a general rule, all carbohydrates in biological systems are D.

Just as the cell can recognize the difference between D and L, the expectation for this course is that you are able to recognize the structures of the carbohydrate but it will not be necessary to know how to draw the structures.

Aldoses

Now we will build larger aldoses by adding one carbon at a time to the structure. In doing this, two components will remain unchanged, the aldehyde group will always be the number one carbon and the bottom or last two carbons will always represent the D form of the carbohydrate (monosaccharide).

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Add another carbon to the structure given above. How many possible structures result from adding another carbon to this carbohydrate? What must remain constant in the representation of these structures?

One of these structures is very important to the biological system: D-ribose. This aldose makes up the backbone structure of RNA and a derivative of it, 2-deoxyribose, makes up the backbone of DNA.

Similarly, an additional carbon can be added to the pentoses to form the possible hexoses (six carbon monosaccharides). An expansive tree on structures can be built starting with the simplest aldose. While all of these structures are possible starting with D-glyceraldehyde, the biological system only utilizes a few of these structures. Of the hexoses, the predominant compound is D-glucose, one of the most prevalent sugars in biology. The linear structure of glucose is shown below, along with ribose.

It is useful to provide shorthand notations and alternative ways of representing the carbohydrates. Below are equivalent alternative representations of the glucose and ribose. The form on the right is typically found in books and manuscripts. It should always be remembered that while the structure on the right would appear to represent carbon as having planar bonding, in fact each of the carbons has a tetrahedral bonding structure.

Cyclization of Aldoses

The five (ribose) and six (glucose) membered aldoses have been depicted in their linear form. However, these compounds will spontaneously form 5 member or 6 member ring structures if possible. These are more stable forms of the compounds.

Now let’s represent the ribose structure as it is actually found in solution. As previously mentioned, compounds will spontaneously form 5 or 6 member rings if possible. For aldoses this is possible because of the reactive character of the aldehyde group. For ribose this means that if the oxygen on the number 4 carbon forms a bond with the number one aldehyde carbon and the hydrogen on the hydroxyl shifts to the carbonyl oxygen on the aldehyde the result is a five-member ring referred to as a furanose.

Examination of the result of this transformation, in which the total number of carbons, hydrogens and oxygens between the two structures has not changed, shows that the number one carbon has now become a new asymmetric (chiral) center where it wasn’t before the ring closure. This new chiral center is called the anomeric carbon. You should examine this structure and convince yourself that the four different substituents attached to the anomeric carbon are unique, making it a chiral center.

The drawing below shows not only the possible structures resulting from the ring closure but also shows the more traditional representation of these structures in their ring configuration. In all of these cases the structure is still ribose. The two new structures created by the ring closure create two additional conformations of the number one carbon: alpha with the hydroxyl on the right of the stick structure or pointing down in the ring representation, and beta with the hydroxyl on the left of the stick structure and pointing up in the ring structure. It should also be noted that as represented in the drawing these structures are all in equilibrium with each other and the alpha structure can be converted to the beta structure and visa versa as long as each structure can be converted to the free aldehyde structure.

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The hexoses are capable of forming a six member pyranose ring by the same mechanism. Ring formation in glucose is shown in the diagram below. As with ribose, a new chiral center is formed, giving two possible forms of glucose, alpha and beta.

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Epimers: Monosaccharides that differ by chirality at one position (besides the anomeric carbon) are called epimers. For example, glucose and galactose are epimers of each other. Galactose is one of the monosaccharides that make up lactose, or milk sugar. There are enzyme catalysts capable of inter-converting galactose to glucose by inverting the chiral center. Thus the galactose that is released from lactose can be concerted to glucose and used for energy.

Ketoses

Ketoses
(Definition)
A ketose is a carbohydrate with a carbonyl at the number 2 carbon that is flanked by carbons on both sides. This is a polar, hydrophilic, water-soluble molecule.
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In the same way that the larger aldoses were generated, single carbons can be added to the structure of the dihydroxyacetone and an expansive tree of structures result. If we examine the result of adding a single carbon, the result is the acquisition of an new asymmetric center. In the case of a four carbon ketose, which is formed by the addition of a C(H2O) group below the ketone group in dihydroxyacetone phosphate, the new chiral center can be either D or L. In general, only the D structures of the ketoses are used by biological systems due to bioselectivity.

While it is clear that many ketose structures are possible, only a few are used in biological systems and the focus for this course will be on the hexose D-fructose. The fructose structure has the ketone group as at the number 2 carbon and the D designation based on the chirality at carbon number 5. Applying the principle that compounds will spontaneously form stable ring structures, and using a mechanism similar to that employed for the aldoses, fructose will spontaneously form a five-member ring structure. These structures are depicted below and are in equilibrium with each other through the free ketone structure.

While the monosaccharides can serve directly as sources of energy, as will be seen elsewhere in the course, they are also the building blocks for many molecules that are used for structure, energy storage and signaling. Here we will explore the process by which simple carbohydrates are linked together using a condensation reaction. Time will be spent on understanding this reaction since it is the same reaction that is used to link amino acids together to make proteins and fatty acids to glycerol to made the components of biological membranes.

As described previously the aldehyde form of aldoses and ketone form of ketoses spontaneously form five and six member ring structures called furanoses and pyranoses respectively. This ring closure was an example of an alcohol functional group reacting with the carbon of the carbonyl functional group in either the aldehyde (aldose) or ketone (ketose) group. This is illustrated below. Notice that while atoms are moving from place to place on the structure, there is no net gain or loss of atoms in the closing of the ring to form what is referred to as a hemiacetal. Furthermore, this ring closure is freely reversible, which allows the alpha form of the ring to be in equilibrium with the beta form.

Starting with the hemiacetal (closed ring) structure, the unique, newly created, asymmetric center has a special name: the anomeric carbon. In the structure of glucose given below, the anomeric carbon, which is created by forming the six member (pyranose) hemiacetal structure, is highlighted. You should examine this structure and convince yourself that the four different substituents attached to that carbon are unique .

This anomeric carbon is the target for the formation of a covalent bond between it and potentially any hydroxyl functional group on any other monosacchardie. The covalent bond formed between the anomeric carbon and a hydroxyl group is called a glycosidic bond and the final structure is referred to as an acetal. The equation for the formation of the glycosidic bond is given below. In this reaction, a molecule of water is lost during the combining of the two glucose molecules to form the glycosidic bond. This type of reaction is referred to as a condensation reaction. The reverse of this reaction, requiring the addition of a water molecule, is referred to as a hydrolysis reaction. These combined forward and reverse reactions (condensation and hydrolysis) form the basis for the creation of most of the covalent assemblies in biological systems.

The hydroxyl groups from the 1 carbon and 4 carbon react to produce an α 1,4-glycosidic bond and water.

While all linkages between the anomeric carbon of one carbohydrate and every hydroxyl of a second carbohydrate are possible, it is important to understand, that in biological systems, very little is left to chance, and in fact, every reaction that takes place in a cell is catalyzed by an enzyme. Just as we have stated that bioselectivity dictates that D carbohydrates are used by biological systems, the formation of glycosidic bonds is catalyzed by specific enzymes that direct the formation of specific glycosidic bonds between the anomeric carbon of one defined carbohydrate and a specific hydroxyl function of a defined second carbohydrate. Each resulting disaccharide is a different structure. This is another example of bioselectivity.

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It should also be noted that while the transformation from the alpha to the beta form of an individual carbohydrate at the anomeric carbon is freely reversible and dictated by equilibrium, that freedom of conversion is lost once the anomeric carbon is involved in a glycosidic bond. Furthermore, the condensation/hydrolysis reaction is generally written as an equilibrium; however, the glycosidic (acetal) linkage is very stable and does not spontaneously break (hydrolyze) without the input of energy and the use of a specific enzyme. The stability of the glycosidic bond contributes to the effective use of the polysaccharides in maintaining structure.

Carbohydrates generally exist as di- and polysaccharides used in transport, energy storage, structure and signaling. While the glycosidic bond is always between the anomeric carbon of one carbohydrate and the hydroxyl of another carbohydrate, the number of hydroxyls per monosaccharide and the different orientations of the hydroxyl on the anomeric carbon make the number of potential structures extremely large. Here we will focus on some of the biologically important di- and polysaccharides with an emphasis on the ability to identify and describe the structures not the ability to draw the structures.

Disaccharides

Two of the most common disaccharides are lactose, or milk sugar, and sucrose, or common table sugar. Each is a form of carbohydrate storage and represents an example of a hetero-disaccharide, i.e. disaccharide formed from two different monosaccharides.

In the case of sucrose, the composition is a molecule of glucose and a molecule of fructose. The structure is described as glucose-alpha,beta 1,2-fructose. In this structure it is instructive to identify the anomeric carbon on the glucose and the fructose molecules since they are both involved in the formation of the glycosidic bond.

A third disaccharide that provides a transition to the common polysaccharides is maltose, a homo-disaccharide. The structure is a homo-dimer made up of two molecules of glucose linked by an alpha-1,4 glycosidic bond. Maltose is commonly found as a dietary supplement and naturally is produced as an intermediate breakdown product of starch. As such it also represents the basic repeating unit of the polysaccharides starch and glycogen.

Polysaccharides

Homopolysaccharides: Three major homopolymers found in nature are starch, glycogen and cellulose. All three are made of the same building block (subunit or monomer) – glucose, but they are different in structure and function.

Starch: The structure of starch has two components: amylose and amylopectin. Amylose is a linear polymer of glucose subunits linked end-to-end by alpha-1, 4 glycosidic bonds. Amylopectin has the same backbone polymer structure as amylose, but also contains branches from the backbone linked by alpha -1,6 glycosidic bonds every 20-30 glucose subunits along the amylose backbone. This gives rise to a highly branched structure used in the storage of glucose in plants.

Glycogen is another example of a homo-polysaccharide of glucose with a repeating structural unit of glucose. For glycogen, there is no linear form, but there is a highly branched form that resembles amylopectin with branch points separated by only 8-10 glucose subunits along the backbone. This creates a much denser, more highly packed structure used for storage of glucose in mammalian cells.

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Cellulose plays a structural role as the key component of a plant cell wall. Cellulose is an unbranched polymer, analogous to amylose; however, the linkages between the glucose subunits are beta-1,4, rather than alpha-1,4. This difference in configuration of glycosidic bond leads to differences in structure and in function. Beta-1,4 linkage results in the linear and extended shape of the polymer structure, which allows the strands of cellulose to align and form a hydrogen-bonding network between the hydroxyls on the individual glucose subunits. This hydrogen bonding adds to the rigidity of cellulose cell wall.

Another demonstration of bioselectivity is at play in the discrimination between cellulose and amylose by our digestive system. The structural difference between the beta-1,4 link in cellulose and the alpha-1,4 glycosidic bond in amylose is sufficient to require different enzymes to break (hydrolyze) the bonds separating the glucose subunits in each structure. The difference is significant because it prevents humans from being able to use cellulose as a glucose source, while we are able to use glycogen and starch. Humans do not posses the enzyme that hydrolyzes the beta-1,4 link in cellulose.

Hetero-Polysaccharides: An enormous set of possibilities for structures exist if one considers that variations can occur between the orientation of the glycosidic bond (alpha vs. beta), the sites to which the bond can be made on an adjacent monosaccharide, the number of different carbohydrates involved (glucose, galactose, ribose, etc), and the order in which they appear.

Such variation, even when a few monosaccharides are included, can give rise to an enormous number of uniquely recognizable structures. In some cases these varied structures can be attached to proteins and lipids to be used as identification/signaling devices on the surface of cells. Each cell type and each species of microorganism can display a unique identification on its surface to be used in cell recognition and to identify partners in cell-cell interactions.

Many other hetero-polysaccharides exist to carryout a variety of functions within an organism. A unique hetero-polysaccharide produced by bacteria is the peptido-glycan that forms the basic structure of the bacterial cell wall. In this case there is a linear hetero-polysaccharide that acts much like the linear strands of cellulose in the plant cell wall. However, as implied by the name, the peptido-glycan is a covalent complex between a polysaccharide (glycan) and a peptide (a fragment of a protein). In the bacterial peptido-glycan, the linear hetero-polysaccharide chains are linked together by covalent bonds using a peptide as the linker. As seen in the illustration below, this means that the bacterial cell wall is fully connected by covalent bonds while the plant cell wall is stabilized by hydrogen bonding between hydroxyl groups on parallel strands.

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Introduction

Amino acids are the building blocks of proteins. The sequence of amino acids in individual proteins is encoded in the DNA of the cell. The physical and chemical properties of the 20 different, naturally occurring amino acids dictate the shape of the protein and its interactions with its environment. Certain short sequences of amino acids in the protein also dictate where the protein resides in the cell. Proteins are composed of hundreds to thousands of amino acids. As you can imagine, protein folding is a complicated process and there are many potential shapes due to the large number of combinations of amino acids. By understanding the properties of the amino acids you will get an appreciation for the limits of protein folding and how to predict the potential higher order structure of the protein.

The numbering of the carbon atoms in an amino acid follows the nomenclature used for carboxylic acids. The first carbon adjacent to the carboxyl group is the α-carbon, followed by the β-carbon, etc. as illustrated in the diagram to the right. All amino acids that are found in proteins have the same backbone structure, the acidic carboxylic acid group, the α–carbon, and an amino group that is attached to the α-carbon, hence the name α-amino acids. The sidechain, or R, group is attached to the α-carbon and is different for each amino acid. Note that the α proton is often not drawn, but its existence should be inferred from the fact that carbon forms four single bonds.

The amino group has a pKa value of ~9, thus it is protonated at pH 7.0. The carboxylic acid group has a pKa of 2.0, and thus it is deprotonated at pH 7.0. Each of the 20 amino acids has a different sidechain(R group), which is attached to the α–carbon. Use the exercise below to identify these important functional groups in an amino acid.


General structure of an α-amino acid. The mainchain atoms, which are found in all amino acids, are highlighted. The remaining carbon atoms (β, γ, etc) form the sidechain of the amino acid. These differ from one amino acid to the next.

Chirality: Because there are four different groups attached to the central carbon, the α–carbon is an asymmetric or chiral center. The chiral center gives rise to D and L enantiomers for each amino acid. However, where bioselectivity dictated the dominance of D carbohydrates, the same discrimination process for amino acids gives rise to the dominance of the use of L amino acids in nature.

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If all of the amino acids have the same basic structure with an amino, a carboxyl and a hydrogen fixed to the α–carbon, then the large variation in the properties and structure of the amino acids must come from the fourth group attached to the α–carbon. This group is referred to as the side chain of the amino acid or the R group. The structures of the 20 common amino acids are shown below. Clicking on the amino acid in the table, displays three-dimensional model of that amino acid below the table.

The sidechain groups of these amino acids contain many of the same functional groups that were discussed in the first module and can be found in the Functional Groups. To learn more about the amino acids, watch the video of Dr. Bill Brown's lecture on amino acids. It may be helpful to review the Functional Groups by clicking the Learn-By-Doing link below.

Peptide Bond

Proteins are polymers of amino acids. The amino acids are joined together by a condensation reaction similar to that described for the formation of the glycosidic bond in polysaccharides. Each amino acid in the polymer is referred to as a residue. Individual amino acids are joined together by the attack of the nitrogen of an amino group of one amino acid on the carbonyl carbon of the carboxyl group of another to create a covalent peptide bond and yield a molecule of water as shown below.

Peptide bond formation occurs by a dehydration reaction. The amino group of the second amino acid attacks the carbonyl carbon of the first, forming the peptide bond and releasing water. The resultant dipeptide has an amino terminus (left) and a carboxy-terminus (right). The mainchain atoms, which are the same for each residue in the peptide, include the nitrogen and its proton, the α–carbon and its hydrogen, and the C=O group. The R-groups form the sidechain atoms.

The resulting peptide chain is linear, defined by the mechanism that builds the polymer, and has defined ends. Short polymers (< 50 residues or amino acids) are usually refereed to as peptides, and longer polymers as proteins. Because the synthesis takes place from the alpha amino group of one amino acid to the carboxyl group of another amino acid, the result is that there will always be a free amino group on one end of the growing polymer (the N-terminus) and a free carboxyl group on the other end (the C-terminus). Note that the potential exists for the formation of amide (peptide) links involving the carboxyl and amino groups in the side chains, but bioselectivity directs the synthesis to be linear, involving only the alpha amino and alpha carboxyl groups.

Note that after the amino acid has been incorporated into the protein, the charges on the α–amino and the carboxyl groups have disappeared. Thus the mainchain atoms have become polar functional groups. Since each residue in a protein has exactly the same mainchain atoms, the functional properties of a protein must arise from the different sidechain groups.

By convention, the sequences of peptides and proteins are written with the N-terminus on the left and the C-terminus on the right. The name of the N-terminal residue is always the first amino acid. The name of each amino acid then follows. The primary sequence of a protein refers to its amino acid sequence.

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Protein sequence

A protein is composed of amino acids attached in a linear order. This basic level of protein structure is called it's primary structure and derives from the formation of peptide bonds between the individual amino acids. The order of the amino acids is determined by information encoded in the cell's genes. An example of a protein sequence is shown below where the one letter abbreviations are used for each of the 20 amino acids used in cellular protein synthesis.

Amino acid sequence of Human Estrogen Receptor
Amino acids are indicated using the single letter code. For example, the amino acid glycine is abbreviated with the letter G.

Higher order structure is determined by the Primary Structure

Proteins do not exist as linear threads in the cells but rather as spontaneously folded higher order structures. The higher order structure is determined by the amino acids in the primary structure. The function of a protein is determined by its higher order structure.

Proteins take on structure in stages defined by the interactions of amino acids with each other in the primary sequence. The higher order stages of secondary, tertiary, and quaternary structure of the estrogen receptor are shown in the movie below. Although the exact structure of the protein was determined using biophysical methods employing sophisticated instruments and calculations, the ability to predict the three dimensional structure (tertiary structure) of a protein de novo is not yet possible. It is possible to make excellent predictions of secondary structure from primary structure and the use of measured constraints on structure based on the structure of the peptide bond and characteristics of individual amino acids is making it possible to approach predictive methods.

The stages or levels of protein structure are displayed here:

  • Primary Structure: The amino acid sequence of the protein, with no regard for the conformation of the amino acids.
  • Secondary Structure: short range interactions involving only mainchain atoms resulting in α-helices and β-sheets
  • Tertiary Structure: long range interactions resulting in the 3-D Folding of a single polypeptide chain.
  • Quaternary Structure: The interaction of two or more peptide chains to make a functional protein. The oxygen transport protein hemoglobin shows this level of structure. The functional protein is composed of four chains.

Constraints Determining Secondary Structure

Peptide Bond is Planar

When two amino acids are joined a peptide bond is formed through a condensation reaction. The carbonyl electrons are partially shared by the carbon nitrogen peptide bond giving the carbonyl carbon/amide nitrogen bond a slight double bond character. Because of this partial double bond character, rotation around the C-N peptide bond is prevented and thus the peptide bond is planar. This is the first and a major constraint placed on protein folding. The figure below depicts the six atoms that are included in the planar peptide bond: the carbonyl carbon, the carbonyl oxygen, the alpha carbon attached to the carbonyl carbon, the amide nitrogen (of the second amino acid), the hydrogen attached to the amide nitrogen, and the alpha carbon of the second amino acid. Two possible orientations of the planer peptide bond are possible, the trans from and the cis form. Although both are planer, the trans form is more stable and this form is shown in the diagram below.

Since there is no rotation around the C-N bond of the peptide bond, the only possible freedom of rotation in an amino acid residue is the carbonyl carbon-alpha carbon single bond, which is denoted as psi(Ψ), and the amide nitrogen-alpha carbon single bond, which is called phi(Φ). If one knows all of the phi-psi angles of rotation for every amino acid residue in a protein, it is possible to define the secondary structure of that protein. It should be noted that not all phi-psi angles are possible due to steric interference, electronic repulsion by side chains of the same charge and other factors all of which represent constraints in the folding of a protein.

Explaining Φ and Ψ

Watch this short video for a description of the Φ and Ψ angles.

Side Chain Constrains Φ and Ψ Angles

Secondary structure is generally defined by the interaction of amino acids adjacent to each other in the primary sequence. Elements of secondary structure include the α-helix, the beta-sheet and the beta-turn. These structures involve short range interactions that involve hydrogen bonding between peptide bonds and do not involve the interactions of amino acid side chains.

Alpha Helix One of the constrained pairs of Φ-Ψ angles gives rise to the secondary structural element referred to as the α-helix. The α-helix is the prominent structure seen hemoglobin shown previously. Because of the regular nature of the structure, every Φ-Ψ angle pair in an α-helix is the same. α-helical structure is stabilized by hydrogen bonding between the backbone carbonyl oxygen and the amide hydrogens. In α-helices there are 3.7 amino acids per turn and the carbonyl of the n-th amino acid hydrogen bonds to the amide hydrogen of the n+4 amino acid. The hydrogen bonds are parallel to the long axis of the helix and the side chains extend to the outside of the helical cylinder.

Beta-Sheet The second predominant secondary structure element is the β-sheet. When the Ψ and Φ angles are close to 180 degrees the peptide chain is fully extended, the β-strand conformation exists. A β-sheet forms when several β-strands run either parallel or anti-parallel to each other. The structures are stabilized by hydrogen bonds between the strands and the anti-parallel structure appears to be more stable due to maximization of the number of possible hydrogen bonds. The atoms participating in hydrogen bonding in a β-sheet are the same as those for α-helices: the carbonyl oxygen and the amide hydrogens. In the case of a β-sheet the hydrogen bonds are in the plane of the sheet and perpendicular to the peptide chain. The side chains project above and below the plane of the β-sheet.

Proline Kinks the System

Proline is unusual among the amino acids and is technically referred to as an imino acid. It is a cyclic molecule forming a five member ring that closes the linear hydrocarbon side chain with the α-amino group. Because of this there is no rotation about the Ψ bond and therefore, proline terminates helix formation and places further constraints on the folding of the peptide chain.

Lecture on Protein Structure

View the following video describing protein structures.

Hydrophobic interactions drive tertiary structure formation

Proteins contain a mix of hydrophobic and hydrophilic amino acids. Hydrophobic molecules contain no or very few highly electronegative atoms and do not form strong bonds with water. In aqueous solutions hydrophobic molecules are driven together to the exclusion of water. This is an example of the hydrophobic effect previously described in the section on bonding. As a protein folds to its final three-dimensional structure, the hydrophobic parts of the protein are forced together and away from the aqueous environment of the cell.

 

Stabilization of the Folded State: The tertiary structure is determined by non-covalent interactions that involve amino acid side chains (side chain and backbone interactions, or side chain-side chain interactions). Amino acid residues involved in these interactions can come from distant parts of the polypeptide chain bringing the chain into a more compact shape. The non-covalent interactions between the side chains include the following:

  1. Iconic bonds
  2. Hydrogen bonds
  3. Hydrophobic/van der Walls interactions

Your should explore the Jmol structure to determine which of the following is more important in stabilizing the tertiary structure.

Disulfide bonds stabilize tertiary structure

Protein folding and stabilization is driven primarily by non-covalent interactions. α-helices and β-sheets are stabilized by hydrogen bonding. Tertiary structure is driven and stabilized by hydrophobic interactions and involves all types of non-covalent bonding. As you will see in the next page, quaternary structure is stabilized primarily by all types of complimentary, non-covalent interactions. One covalent bond is possible and is formed when two cysteine amino acids (sulfhydryl containing side chain) are close together as a result of tertiary structure formation. The sulfhydryl group is highly reactive and will covalently bond with another sulfhydryl group to form a covalent disulfide bond (a cystine residue). Disulfide bonds also form between peptide chains in forming quaternary structure, for example in the structure of antibodies. The disulfide bond is reversible and is sensitive to the environment.

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More than one peptide forms a functional protein

There are many examples of proteins that require more than one peptide chain to be functional. In the case of the estrogen receptor two identical peptide chains come together to form the functional protein. This is called a homo dimer. Hemoglobin is composed of four peptide chains; two identical alpha chains and two identical beta chains. The individual peptides or sub-units of estrogen and hemoglobin are held together by all of the non-covalent bonding types: hydrogen bonding, ionic bonding and hydrophobic interactions .

The interaction of the subunits in a quaternary complex represents another form of equilibrium. The individual polypeptide chains are made separately and must associate through specific complimentary interactions.

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Antibodies represent a different type of quaternary structure because the individual subunits are linked by disulfide bonds. The structure is a dimer of dimers and the basic unit is a large peptide (the heavy chain) bound to a smaller chain (light chain) by disulfide bonds. Since this dimer is a combination of two different peptides it is called a heterodimer. Two of these heterodimers are joined by disulfide bonds between the heavy chains to form the final tetrameric structure.

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Protein - Ligand Interations

In this module we will use all of the concepts previously introduced in the section on biological chemistry to study the interaction, or binding, of small molecules to proteins. If the small molecule binds to a protein, but undergoes no further changes, then it is referred to as a ligand. If the molecule binds to the protein, and then is changed by a protein-catalyzed chemical reaction, the small molecule is referred to as a substrate, the resultant altered compound is the product, and the protein becomes an enzyme. Although ligands and substrates are usually small molecules, proteins themselves can be ligands and substrates for other proteins/enzymes.

Protein-ligand interactions serve several important functions in the cell. They may be responsible for buffering the ligand concentration and the transport of ligands. Oxygen binding to myoglobin is an example of buffering while the protein hemoglobin is involved in the transport of oxygen from the lungs to the tissues.

It is also possible that binding the ligand causes the functionality of the protein to change from active to inactive, or from inactive to active. For example, the estrogen receptor protein, shown below in yellow and purple, binds DNA only when estrogen (red) is bound to the protein. Estrogen is the "key" that turns the estrogen receptor "on." In the figure below, the estrogen receptor is depicted with its secondary structure highlighted. As you can see it is mostly α-helices. The estrogen receptor represents a quaternary structure composed of a dimer of two protein chains shown as yellow and purple in the figure. 17-β-estradiol (an estrogen), the red space-filling balls, fits in a properly sized pocket on each of the proteins in the dimer. Only when both pockets of the dimer are occupied by estrogen is the receptor activated and able to bind to DNA. As you can see the estrogen binding pocket is only a small part of the total protein size.

The estrogen receptor binds to estrogen and this causes estrogen receptor to bind to DNA and turn genes on and off. The result in some cells is the commencement of cell division. When estrogen falls off, estrogen can no longer bind DNA and division may stop. If estrogen did not dissociate from the estrogen receptor then cell division could not be controlled and cancer could result.

Energetics of Protein-Ligand Complex Formation

The binding of ligands and substrates relies primarily on the characteristics of the non-covalent bonds and the functional groups associated with both the protein and the small molecule. The interactions that can be used are:

  1. van der Waals interactions: the protein and its ligand are complementary in shape, optimizing the contact between the two surfaces.
  2. hydrogen bonding
  3. hydrophobic interactions,
  4. ionic bonds

The functional groups that are responsible for these interactions are those found in the side chains of amino acids, the peptide bonds in the protein and the small molecule binding partner. Following some simple rules it is possible to predict how two molecules might interact. In some cases It is also possible to predict how a cell might alter physiological conditions to regulate that binding. You will explore that interactions that stabilize bound estrogen to the estrogen receptor in the following activity:

Ligand Binding is Reversible

Because protein and ligand interactions are non-covalent in nature it is possible for the ligand to dissociate from the protein. This reversible binding is critical and is generally represented as:

Macromolecule (protein) + Ligand <=> [Macromolecule Ligand]

M + L <=> [ML]

The bound complex is surrounded by brackets, [ ], to indicate that the ligand is physically associated with the macromolecule.

In the following pages you will explore reversible binding and how we can determine the strength of the interaction between a protein and its ligand by experimental measurements.

As discussed previously, proteins bind and release their ligands. In this section we will explore the reversible binding and the equilibrium state. We will use myoglobin, as an example, to explore the equilibrium established between the bound protein ligand complex and free reactants. Myoglobin is a single polypeptide chain protein that binds oxygen to create an oxygen buffer in muscle cells. The reversibly binding of oxygen to myoglobin can be expressed as follows:

Myoglobin + Oxygen <=> [Myoglobin Oxygen]
M + O <=> [MO]

By reversibly binding oxygen, myoglobin acts as an oxygen buffer. When free oxygen is high myoglobin binds oxygen and the equilibrium shifts to the right. If the oxygen concentration falls the equilibrium shifts to the left to free oxygen and protein. The change in equilibrium is only possible because protein ligand binding is reversible. The direction of the equilibrium, either towards more complex or towards free reactants, depends primarily on the amount of free oxygen in the cell. Oxygen supply is always changing depending on activity level. Myoglobin supply is essentially constant in the cell since protein synthesis is relatively a much slower process.

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In the Learn By Doing below, you will explore the equilibrium between free protein, ligand, and the complex, and how ligand concentration affects the equilibrium.

learn by doing

The number of the contacts between a protein and its ligand and the environment of the binding pocket determine how tightly the ligand binds to the protein. It is difficult to predict how tightly a ligand will bind to a protein or to predict the structure of ligands that will bind tightly a binding pocket of a known protein. There is intensive research in this area and forms the basis for many computational studies used in drug discovery laboratories. The tightness of the binding is a measurable parameter and describes the affinity of a ligand for a protein.

In the previous section the binding of oxygen to myoglobin was defined by the equation:

M + O <=> [MO]
This equation describes an association of two molecules. In the previous analysis of weak electrolytes we wrote the dissociation of a weak acid as the taking apart of a molecule that was bound by a weak covalent bond. While there are no covalent bonds being formed during the association of oxygen and myoglobin, there is the same type of equilibrium established between the association of myoglobin and the dissociation of the weak acid. Thus, where we used a Kd (K) as a constant that defined the strength of the weak electrolyte's ability to retain its proton, it is also possible to define the affinity of the ligand for the protein by a constant referred to as the equilibrium constant, Keq. For the association reaction as it is written above, the equilibrium constant would be expressed as follows:

Keq=[MO][M]*[O]

In general the affinity of the ligand will increase proportionately as the number of specific interactions between the ligand and the protein increase. In the example of the binding pocket of the estrogen receptor there are several hydrogen bond contacts and a hydrophobic pocket for the mostly hydrophobic estrogen. Other protein ligand binding interactions are based on fewer contacts, as in the case of oxygen binding to myoglobin and thus the affinity is lower.

Previously, you explored how proteins interact with their ligands, using the example of myoglobin and oxygen. Proteins are not only storage proteins, but can also act as hormone receptors as seen with the estrogen receptor and many serve as enzymes that catalyze processes in the cell.

Catalyst
(Definition)
A catalyst is a participant in a chemical reaction that speeds up the reaction but is not consumed itself. Enzymes are biological catalysts that mediate the conversion of substrate to product, by lowering the activation energy of the reaction. Enzymes re not consumed or modified during the process. Enzymes are generally made of proteins but nucleic acid enzymes, ribozymes, have also been discovered.

General Reaction

Cells operate within the laws of chemistry and physics, and utilize only chemical reactions that occur naturally. Many of these reactions are extremely slow, requiring years to eons to complete. For example, your favorite book contains paper made of cellulose from trees. This paper will take decades to degrade in the environment and mammalian systems lack the enzyme that is specific to degrade cellulose, however, a bacteria found in the stomach of cows and other ruminants have the ability to digest cellulose and provide glucose to the cow. For a cow to survive it cannot wait decades to digest a meal of hay. To meet the speed of life, chemical reactions in cells are sped up by enzyme catalysts.

Enzymes bind to substrates in a manner similar to the way myoglobin binds oxygen or the estradiol binds to the estrogen receptor, but enzymes can go one step further. In this case the ligand is specifically referred to as the substrate (the molecule that the enzyme will convert to product) and it binds to a specific binding region of the enzyme referred to as the active site. Once bound, the ligand, or substrate, can either simply reversibly come off the enzyme, or it can be converted into a new compound or product. The general form of the reaction is:

E + S <=> [ES] -> P + E
Where E = enzyme, S = substrate, ES = the enzyme•substrate complex, and P = product. The square brackets around the ES complex indicate that the complex is inferred, because it is very unstable and generally not directly measurable.

Catalysts Lower Activation Energy

Enzymes and other catalysts lower the activation energy for converting a specific substrate to product, as shown in the graph below. The activation energy is the energy required for a chemical reaction to proceed to product. By lowering the activation energy, a chemical reaction can proceed much more quickly. Note that enzymes have no effect on the free energy of the reaction. Enzymes are not consumed in the process, but are recycled for further catalysis.

Catalysts Lower Activation Energy
This graph shows how enzymes catalyze reactions by lowering the activation energy. Without a catalyst, the amount of energy required for the reaction is large. By fixing a substrate in its active site, enzymes lower the activation energy, increasing the rate of the reaction.

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The Rate of Product Formation.

In the last section, the action of an enzyme was described as taking place in two parts. The first part involves the binding of substrate to enzyme in a reversible equilibrium as modeled in the way of ligand binding. The second part involves the conversion of the [ES] complex to product and the release of the product from the enzyme. This step represents the catalysis step in the enzyme catalyzed reaction. These parts are illustrated in the overall reaction below:

E + S <=> [ES] -> P + E

The rate, or velocity (v), of the reaction is usually determined by measuring the amount of product (P) produced/unit time:

v=dP/dt

The dependence of the reaction velocity on the kinetics of substrate binding, substrate release, and conversion to product is difficult to calculate under all conditions. However, if steady-state conditions are assumed then a very simple relationship can be found. Steady-state conditions imply that during the measurement the concentrations of the various species, e.g. [ES] ,do not change. Under steady-state conditions the following equation describes the relationship between the measured rate (v) of the reaction and the initial concentration of substrate (S) and the concentration of the enzyme. This equation is the Michaelis Menton-Briggs Haldane equation.

v(Et,[S])=kCATEt[S]KM+[S]=VMAX[S]KM+[S]

In this equation, kcat is the rate at which the [ES] complex decays to product. Vmax is kcat multiplied by the total amount of enzyme, Et. It is therefore the maximum velocity at which the reaction can occur under a fixed enzyme concentration. Km is also a constant that reflects the affinity of the enzyme for the substrate and is an approximation of the dissociation equilibrium constant. Both kcat and Km depend on the particular enzyme and substrate.

This equation can also be represented graphically by the hyperbolic curve illustrated below. This curve can be dissected into two parts: a region at the beginning where the velocity of the reaction increases proportionately with the amount of substrate present initially in the reaction, and the later region of the curve, at high substrate concentration, where the rate of the reaction is independent of the substrate concentration. This latter region defines saturation of the enzyme by substrate and the velocity approaches the value of Vmax.

Effect of substrate concentration on enzyme velocity.
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Enzyme catalysis is affected by many factors including substrate and enzyme concentrations, pH, temperature, and the presence of inhibitors. The learn-by-doing on this page will explore the effect of substrate concentration. The following page will explore the effect of enzyme concentration, pH, temperature, and inhibitors on the velocity of the enzyme catalyzed reaction.

Enzyme Catalysis

This simulation will demonstrate the effect of substrate on the reaction velocity.

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Left alone, enzymes would deplete a cell of all the substrates. In some cases, this may be desirable, such as enzymes that degrade old, worn-out proteins. In other cases, there may be multiple uses for a substrate and depletion, by one enzyme, may be detrimental to the working of a cell. In some cases, such as the digestion of food, the enzyme needs to be functional in the gut but not in the cell. Thus, several types of regulations have evolved that the cell can use to control enzyme rate.

Control by Enzyme Concentration

A common way of regulating enzyme activity is by increasing or decreasing the amount of enzyme by regulation of the rates of synthesis in the cell. If you recall, the rate of reaction is proportional to the concentration of the enzyme-substrate complex ([ES]). The concentration of this complex can be increased in two ways: i) either by increasing [S], which you explored on the previous page, or ii) by increasing the total amount of enzyme in the cell.

Control by Temperature

One mechanism to control the rate of product formation is through temperature. Velocity of product formation increases linearly as the temperature increases until a critical temperature is reached as indicated in the following graph.

Velocity of Product formation versus temperature.

Product formation ceases beyond the critical point due to two effects of temperature. One effect is to decrease the non-covalent interactions between enzyme and substrate. Remember that ionic and hydrogen bonds are weak non-covalent bonds representing 5 Kcal/mole each.only 5 kcal/mol in strength. When the temperature increase matches the energy of the bond, the energy cn be used to break the bond. Thus if the enzyme cannot bind the substrate, no product is formed.

The second effect of temperature is to cause proteins to denature (unfold). Denaturation means that the protein loses the secondary, tertiary, and quaternary structure required for function. As discussed earlier, the protein tertiary structure is stabilized by non-covalent bonding. As with substrate binding, increased temperature can provide the energy necessary to break the weak non-covalent bonds. In some cases, the beaking of a single hydrogen bond can alter the structure of the enzyme sufficiently to cause the loss of function (i.e. loss of the ability to bind substrate, to recognize the substrate or to catalyze product formation).

Control by pH

The pH of the environment a protein finds itself in can drastically affect the proteins function and denaturation state. Have you ever put lemon and cream into hot tea at the same time? The cream curdles because the low pH of the lemon juice causes the proteins to denature. pH also affects the ability of a protein to bind to its ligand. At low pH hydrogen bond acceptors such as carboxyl groups become protonated. If they are protonated then they cannot form the critical ionic bonds between the protein and the ligand. The graph below shows the activity of a typical protein as a function of pH. This protein is most active around neutral pH.

The cell can use pH as a mechanism to control enzyme action. Enzymes used in digestion that destroy other proteins could be detrimental to a cell. However, typically these enzymes are only active at low pH such as is found in the stomach or in intracellular compartments such as the lysosome that is maintained at lower pH than the cytoplasm.

In vivo Control - Competitive and Non-competitive Inhibition

Temperature and pH represent environmental means of regulating the velocity of an enzyme catalyzed reaction. However, in a typical organism (in vivo) the temperature and pH are usually regulated and therefore cannot be used to control enzyme activity. Consequently, the velocity of enzymatic reactions are usually regulated by molecular interaction of a second molecule with the enzyme. These are generally termed inhibitors if the binding of the second molecules causes a loss of enzyme activity (reduced velocity at any given substrate concentration).

In competitive inhibition the second molecule binds in the active site thus preventing the substrate from binding. The regulatory molecule competes with the substrate for the active site. This decreases the rate at which the enzyme can convert the substrate to product because the enzyme spends part of the time unable to bind the substrate. This can be seen in the graph below plotted as the dashed red line. The occupancy of the binding pocket is in equilibrium between the binding of the substrate or the inhibitor. As you can see from the curve the maximum velocity is unchanged, but the substrate concentration required to reach maximum velocity is increased.

Non-competitive inhibition involves a second small molecule binding to the enzyme. In this case there are two binding sites: one for the substrate and a distinct one for the inhibitor. When the inhibitor binds to the enzyme, the substrate binding pocket changes such that the substrate is no longer able to bind. It may also be possible for both the substrate and inhibitor to bind the enzyme at the same time but the enzyme is unable to complete the conversion of substrate to product. Non-competitive inhibition results in a lower maximum velocity for the enzyme when the inhibitor is present as can be seen in the green curve. Inihibitors of this type are often referred to as allosteric inhibitors because they cause the shape (steric) of the enzyme to assume a different (allo) form.

Introduction to Lipids

The cell is composed of two distinctive environments: the hydrophilic aqueous cytoplasm and the hydrophobic lipid membranes. The lipid environment is defined by the family of molecules that are characterized by their hydrophobic nature and their common metabolic origin. Three members of the lipid family of molecules will be discussed in this course: fats (triacylglcerol), phospholipids, and steroids.

The Structure of Lipids

Lipid molecules are slightly soluble to insoluble in water. Lipids are hydrophobic because the molecules consist of long, 16-18 carbon, hydrocarbon chains (or backbones) with only a small amount of oxygen containing groups. Lipids serve many functions in organisms. Fats (or triglycerides) are used to store energy. Phospholipids and steroids are the key components of cell membranes. Lipds are also the major components of waxes, pigments, and steroid hormones.

Fats (triacylglycerols, triglycerides)

Various forms of a 12-carbon fatty acid are shown. The top structure is the fully saturated fat, lauric acid. The two lower structures show two forms of unsaturated fatty acids that can be generated from lauric acid by formation of a single double bond. The cis (left) form is typically found in biological lipids, note that the double bond has kinked the hydrocarbon chain. The trans (right) form is generated by a chemical process that transforms unsaturated fatty acids to saturated fatty acids. This process is used to convert liquid vegetable oils to solid margarine.

Fats are synthesized from two different classes of molecules: fatty acids and alcohol. The fatty acids are unbranched hydrocarbons that terminate with a single carboxyl functional group. The fatty acids are generally 16-22 carbons long and can be both saturated and unsaturated. Saturated fatty acids have no carbon-carbon double bonds (they are saturated with hydrogen), while the unsaturated fatty acids have one to three double bonds along the backbone carbon chain. These double bonds introduce "kinks" in the carbon chain which have important consequences on the fluid nature of lipid membranes. Unsaturated fatty acids have lower melting points than saturated fatty acids.

To construct a fat, or triacylglycerol, three fatty acid molecules are attached to the glycerol through an ester bond between the carboxyl group of the fatty acids and the three alcohol groups of a glycerol molecule. This is another example of a condensation reaction that results in formation of an ester in this case and the release of a water molecule. A fat molecule can be composed of one, two, or three different types of fatty acids each of which can be saturated or unsaturated.

An unsaturated fat has at least one unsaturated fatty acid, whereas a saturated fat has none. Because the double bonds of the unsaturated fatty acids introduce kinks in the hydrocarbon backbone, unsaturated fats will not pack into a regular structure and thus remain fluid at lower temperatures. A saturated fat will pack well and be a solid a low temperatures.

Fats are mainly energy storage and insulating molecules. Per gram, fats contain twice as much energy as carbohydrates. Layers of fat also surround the vital organs of animals to cushion them, and layers of fat under the skin of animals provide insulation.

Phospholipids

Phospholipids contain only two fatty acids attached to a glycerol head. This occurs by a condensation reaction similar to the one discussed above. The third alcohol of the glycerol forms an ester bond through reaction with phosphoric acid. This is another example of a condensation reaction between an acid and an alcohol with the release of water. As a triprotic acid (i.e it has three acidic functions on the phorphorus atom) the phosphate group attached to the glycerol has the potential to form ester links with a variety of other molecules such as carbohydrates, choline, inositol and amino acids. The phosphate group along with the glycerol group make the head of the phospholipid hydrophilic, whereas the fatty acid tail is hydrophobic. Thus phospholipids are amphipathic:a molecule with a polar end and a hydrophobic end. When phospholipids are in an aqueous solution they will self assemble into micelles or bilayers, structures that exclude water molecules from the hydrophobic tails while keeping the hydrophilic head in contact with the aqueous solution. View the animation that demonstrates the formation of micelles and bilayers.

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Click the green arrow to play the animation.
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Phospholipids serve a major function in the cells of all organisms: they form the phospholipid membranes that surround the cell and intracellular organelles such as the mitochondria. The cell membrane is a fluid, semi-permeable bilayer that separates the cell's contents from the environment, see animation below. The membrane is fluid at physiological temperatures and allows cells to change shape due to physical constraints or changing cellular volumes. The phospholipid membrane allows free diffusion of some small molecules such as oxygen, carbon dioxide, and small hydrocarbons, but not charged ions, polar molecules or other larger molecules such as glucose. This semi-permeable nature of the membrane allows the cell to maintain the composition of the cytoplasm independent of the external environment.

A closer view of a Lipid Bilayer forming a membrane

Steroids

The steroids are a family of lipids based on a molecule with four fused carbon rings. This family includes many hormones and cholesterol. Cholesterol is a component of the cell membrane in animals and functions to moderate membrane fluidity because it restricts the motion of the fatty acid tails.

Structure of Cholesterol
Cholesterol in the membrane decreases the fluidity.

Review of Lipids

Examine the effect of cholesterol on membrane fluidity using the animations below.

Use the play/pause button on the left to start or stop the animation. Use menu to move between scenes.
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The Structures of the Cell Membrane

Fluid Quality of Membranes

The cell membrane must be a dynamic structure if the cell is to grow and respond to environmental changes. To keep the membrane fluid at physiological temperatures the cell alters the composition of the phospholipids. The right ratio of saturated to unsaturated fatty acids keeps the membrane fluid at any temperature conducive to life. For example winter wheat responds to decreasing temperatures by increasing the amount of unsaturated fatty acids in cell membranes. In animal cells cholesterol helps to prevent the packing of fatty acid tails and thus lowers the requirement of unsaturated fatty acids. This helps maintain the fluid nature of the cell membrane without it becoming too liquid at body temperature. The fluidity of the membrane is demonstrated in the following animation. The lipids in the membrane are in random bulk flow moving about 22 µm (micrometers) per second. Phospholipids freely move in the same layer of the membrane and rarely flip to the other layer. Flipping of phospholipids from one layer to the other rarely occurs because flipping requires the hydrophilic head to pass through the hydrophobic region of the bilayer.

Click the green arrow to play the animation.

The Mosaic Quality of Membranes

Proteins

Because the cell membrane is only semipermeable, the cell needs a way to communicate with other cells and exchange nutrients with the extracellular space. These roles are primarily filled by proteins. Membrane proteins are classified into two major categories, integral proteins and peripheral proteins.  Integral membrane proteins are those proteins that are embedded in the lipid bilayer and are generally characterized by their solubility in non-polar, hydrophobic solvents. Transmembrane proteins are examples of integral proteins with hydrophobic regions that completely span the hydrophobic interior of the membrane. The parts of the protein exposed to the interior and exterior of the cell are hydrophilic. Integral proteins can serve as pores that selectively allow ions or nutrients into the cell. They also transmit signals into and out of the cell. Unlike integral proteins that span the membrane, peripheral proteins reside on only one side of the membrane and are often attached to integral proteins. Some peripheral proteins serve as anchor points for the cytoskeleton or extracellular fibers. Proteins are much larger than lipids and move more slowly. Some move in seemingly directed manner while others drift.

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Test My System

Carbohydrates

The extracellular surface of the cell membrane is decorated with carbohydrate groups attached to lipids and proteins. Carbohydrates are added to lipids and proteins by a process called glycosylation, and are called glycolipids or glycoproteins. These short carbohydrates, or oligosaccharides, are usually chains of 15 or fewer sugar molecules. Oligosaccharides give a cell identity (i.e., distinguishing self from non-self) and are the distinguishing factor in human blood types and transplant rejection.

Membranes are Asymmetric

As discussed above and seen in the picture, the cell membrane is asymmetric. The extracellular face of the membrane is in contact with the extracellular matrix. The extracellular side of the membrane contains oligosaccharides that distinguish the cell as self. It also contains the end of integral proteins that interact with signals from other cells and sense the extracellular environment. The inner membrane is in contact the contents of the cell. This side of the membrane anchors to the cytoskeleton and contains the end of integral proteins that relay signals received on the external side.

Summary: Membranes as Mosaics of Structure and Function

The biological membrane is a collage of many different proteins embedded in the fluid matrix of the lipid bilayer. The lipid bilayer is the main fabric of the membrane, and its structure creates a semi-permeable membrane. The hydrophobic core impedes the diffusion of hydrophilic structures, such as ions and polar molecules but allows hydrophobic molecules, which can dissolve in the membrane, to cross it with ease. Proteins determine most of the membrane's specific functions. The plasma membrane and the membranes of the various organelles each have unique collections of proteins. For example, to date more than 50 kinds of proteins have been found in the plasma membrane of red blood cells.

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Membrane Transport

The cell membrane provides a semi-permeable barrier between the inside and the outside of the cell. This barrier provides control for transport of nutrients, ions and signals between the highly variable outside environment and the relatively well-defined interior of the cell. This unit of the course will explore the ways in which molecules can pass across the membrane (diffusion and active transport), can be transported into the cell without passing across the membrane (endocytosis), and can send signals for actions within the cell without actually passing across the membrane themselves (signal transduction).

Passive/Simple Diffusion:

Both large and small molecules follow the same general principal of diffusion. Molecules spontaneously move from areas of high concentration to areas of low concentration following Brownian motion. The classic example is the diffusion of a drop of ink placed in a beaker of water. The concentrated drop of color slowly disperses (diffuses) until at some point equilibrium is reached in which the beaker appears to have a uniform color. The following animation depicts this simple diffusion process. Add ink to each beaker and watch the diffusion process. After a period of time, is there a difference in the distribution of ink in each beaker? Follow the yellow ink molecule in each beaker for some time. In which simulation does the yellow ball take longer to transverse the beaker?

Question: If this simulation represented the movement of the ink in air (no gravity considerations), would the movement be faster or slower in water? Why?

The introduction of a cell or liposome into the solution places a barrier to the molecules. As three different molecules diffuse to equilibrium they encounter the lipid bilayer depicted by the horizontal membrane across the center of the stage in the following animation. Note that one type of molecule passes freely through the lipid bilayer while the second type of molecule only occasionally passes through the membrane and the lipid bilayer is totally impermeable to the third type of molecule.

Of the first two types of molecules, the first type might include a molecule such as cholesterol which has some solubility in water but is highly soluble in the non-polar environment of the lipid bilayer and thus will freely pass into the hydrophobic environment of the membrane, distribute freely in the membrane and then some fraction will dissolve in the aqueous environment of the cytoplasm. A second example of this type of permeable molecule is water which while polar is small and able to freely pass across the membrane. The lipid bilayer is much less permeable to the second type of molecule indicating a more polar character and a larger size. Examples of such molecules are the sodium and chloride ions. As a general rule, charged molecules are much less permeable to the lipid bilayer. The third type of molecule is very polar, in many cases charged, and usually a larger molecule. Examples are carbohydrates and amino acids.

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Aspirin
In Protonated and non-Protonated States

Osmosis

Cells continually encounter changes in their external ionic environment and will spontaneously respond by attempting to equalize the concentration of ions on the inside and outside of the cell. Because the plasma membrane (lipid bilayer) is significantly less permeable to ions than water, the establishment of an equal concentration of the ions on either side of the membrane is accomplished by the net movement of water toward the higher concentration of ions to reduce the concentration. This movement of water in response to an imbalance of solute (ion) is referred to as osmosis. This is illustrated in the following simulation.

learn by doing
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Three different conditions may exist in the relationship between the solute (ion) concentration and solvent (water) concentration across a membrane. Isotonic, hypertonic and hypotonic refer to the relative concentration of the solute (small molecules) in the extracellular (outside) space surrounding the cell relative to the solute concentration inside the cell. In an isotonic solution, the concentration of the solute and therefore solvent water (water potential) is the same on both sides. A hypotonic solution is one whose solute concentration is lower (water concentration is higher [i.e. high water potential]) in the extracellular space than inside the cell. Because the water (the solvent) can more easily pass through the membrane than can the solute (ions), the net flow is spontaneous in the direction of the solvent (water) moving from its higher concentration (high water potential) outside the cell to the inside of the cell. Conversely, a hypertonic solution refers to an extracellular solution with a higher solute concentration (lower water concentration [i.e. low water potential]) outside the cell than inside the cell. In this case, the more permeable solvent, water, would flow spontaneously out of the cell toward the low water potential to dilute the solute molecules and create an equal concentration of solute molecules on both sides of the membrane.

Facilitated Diffusion

Cells must be able to move polar molecules such as nutrients and ions across the lipid bilayer of the membrane in order to carry out life processes. But the molecules will still move spontaneously down a concentration from high to low concentration. To allow the polar molecules, which are not soluble in the lipid bilayer, to pass across the hydrophobic barrier it is necessary to provide ports, channels or holes through the membrane. These channels can either allow the molecules to move freely according to their concentration differences or they can be gated channels that control the movement of the polar molecules according to the needs of the cell. In most cases these channels are very discriminatory and will only allow specific molecules to pass; another example of bioselectivity. The channels facilitate the movement of molecules that otherwise would not be spontaneously permeable to the lipid bilayer. The process of moving impermeable molecules across a membrane using channels or pores is referred to as facilitated diffusion. Because the molecules are moving down a concentration gradient the process is driven by simple diffusion. The following simulation depicts the facilitated diffusion of glucose across the membrane using the glucose permease transporter.

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In some cases it is necessary to move molecules against a gradient. The eukaryotc cell, a typical mammalian cell, has many compartments within the cell each surrounded by a lipid bilayer membrane. In most cases the environment within the compartment is different than that in the cytoplasm. An example is the lysosome, a degradative organelle (membrane bound compartment within the cell) whose function is to digest macromolecules delivered either from the outside of the cell or from other compartments within the cell. To carry out this function the lysosome maintains a much lower pH inside the lysosome relative to the cytoplasm. At equilibrium, the concentration of protons would be equal on both the inside and outside of the lysosome.

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To decrease the pH inside of the lysosome, the concentration of protons will need to be greater inside the lysosome than in the cytoplasm. To accomplish this protons will need to move from a low concentration to a high concentration. This is a non-spontaneous process and requires the cell to do work to move the ions up-hill against the gradient. To do work, the cell must expend energy and actively move (pump) the ions. This process is referred to as active transport. The source of energy for this process in most biological systems is the hydrolysis of ATP.

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The following animation depicts another example of active transport; the sodium-potassium ATPase. This active transport system moves three sodium ions out of the cell and two potassium ions into the cell, each against a gradient.

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Transport Proteins

Facilitated diffusion and active transport both require channels or ports in the membrane through which the generally non-permeable molecules can pass. These protein transporters contribute to the mosaic character of the fluid mosaic character of the biological membrane. There are a variety of different structures associated with transport proteins and at the same time many transport proteins that carry out similar functions (e.g. ion channels) have structural similarities while maintaining their ability to discriminate between molecules. Thus transport proteins have been classified both by structure and by function. For the purposes of this course, the classification will be that of function though similarities in structure will be observed in the examples chosen.

There are three classifications of transport proteins based on mechanism of transport: Uniport, Symport and Antiport. The animations on the following pages will demonstrate the three classes of proteins with examples of each.

Transport Proteins

Facilitated diffusion and active transport both require channels or ports in the membrane through which the generally non-permeable molecules can pass. These protein transporters contribute to the mosaic character of the fluid mosaic character of the biological membrane. There are a variety of different structures associated with transport proteins and at the same time many transport proteins that carry out similar functions (e.g. ion channels) have structural similarities while maintaining their ability to discriminate between molecules. Thus transport proteins have been classified both by structure and by function. For the purposes of this course, the classification will be that of function though similarities in structure will be observed in the examples chosen. There are three classifications of transport proteins based on mechanism of transport: Uniport, Symport and Antiport. The following image illustrates the three classes of proteins with examples of each.

Symport

A symport transports two different molecules across the membrane in the same direction in a cooperative manner.

Click the green arrow to play the animation.

Antiport

Antiports transport two different molecules through the membrane in opposite directions.

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Endocytosis

While facilitated diffusion and active transport account for a great deal of the specific uptake of molecules and ions needed by the cell, other sources of external matter can also be taken up by the cell. The channels and pores provide a means by which molecules can pass directly through the membrane to the cytoplasm. Other mechanisms also exist by which molecules are taken up by the cell but do not directly pass through the plasma membrane. This mechanism is referred to as endocytosis. This mechanism involves the engulfing of the matter by the plasma membrane and internalization of the engulfed material inside a cytoplasmic vesicle. Non-specific uptake of molecules occurs by phagocytosis (large particles and macromolecules) and pinocytosis (water soluble small molecules).

In general endocytosis, a molecule or particle encounters the cell surface and randomly causes the membrane to create first a pit in the membrane, followed by a further invagination of the plasma membrane and finally the pinching off of the plasma membrane around the molecule or particle resulting in the formation of a vesicle in the cytoplasm of the cell. Note that during the process the asymmetry of the membrane would be apparent by the fact that the inside surface of the newly formed vesicle is the same as the exterior surface of the cell. Thus the integrity of the cytoplasm and its exposure to only the inside surface of the membrane is preserved. Exocytosis is just the reverse of this process with the fusion of an internal vesicle with the plasma membrane thus releasing its content to the outside. The balance of exocytosis and endocytosis preserves the size of the plasma membrane and ensure neither growth nor shrinking of the cell size.

Once internalized the new vesicle fuses with a slightly acidic early endosome and subsequently with the lysosome where the contents of the original endocytic vesicle are digested and the digested products released to the cytoplasm where they are available for use by the cell. This process is depicted in the following animation.

You can use the magnifier in the lower righ-hand corner to zoom the animation

Protein Transduction – an example of macropinocytosis

In the early 1990’s an intriguing observation was made that has lead to a number of new approaches to drug delivery and therapeutic delivery systems. It is based on an observation of the activity of the transactivator TAT protein associated with the human immunodeficiency virus (HIV-1) and subsequently polyarginine (arginine is a positively charge, naturally occurring amino acid) and other proteins containing a basic peptide region referred to as the protein transduction domain (PTD). The observation was the translocation of virtually any molecule, particle, even liposome that has the PTD attached. While there is still some debate as to the exact mechanism of the translocation, there is some agreement on the general process. The highly positively charged PTD, attached to its ‘cargo’, has a tight electrostatic (ionic) interaction with certain molecules, which are ubiquitous to all cells, in the plasma membrane. Binding to the surface initiates macropinocytosis (pinocytosis with a slightly larger soluble molecule). The presence of the PTD provides for high efficiency initiation of endocytosis. While not involving a specific receptor, the binding by the PTD to the cell surface reduces the concentration dependence for initiation of internalization.

You can use the magnifier in the lower righ-hand corner to zoom the animation
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In contrast to the normal endocytic vesicle the PTD directed endocytic vesicle undergoes retrograde transport. In this process the new vesicle fusses with the Golgi and is the PTD containing proteins are transported back to the Rough Endoplasmic Reticulum where they undergo post-translational modification and are transduced directly to the cytoplasm.

Receptor Mediated Endocytosis

Much as the channels and pores discriminate between specific molecules and their transport through the membrane, specificity and discrimination are seen during endocytosis using specific membrane bound receptors. This targeting defines the uptake of specific molecules or assemblies by specific cells. The following animation demonstrates the process of receptor mediated endocytosis for the Low Density Lipoprotein (LDL) complex. The process is divided into the individual steps to emphasize similarities and differences among general endocytosis, protein transduction and receptor mediated endocytosis. Several general concepts are illustrated during the process including bioselectivity and intermediate recycling.

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The plasma membrane is a very selective barrier. We have seen how some small molecule pass freely, but most molecules are selectively brought into the cells using transporter proteins. Most of these small molecules are metabolites or ions used in the general metabolism of the cell. The cell also needs to transduce information across its membrane. Cells receive signals from the surrounding fluids and other cells. These signals may tell the cell to divide or prevent division and promote growth.

The animation below demonstrates the action of signal transduction through a G-Protein coupled receptor. The ligand is the external signal and it binds the receptor. The G-Protein complex is now able to bind to the receptor. This activates the G-protein by allowing the exchange of GTP for GDP. When bound to GTP the G-protein is able to bind to Adenylate cyclase and activate it. Adenylate cyclase generates the internal signal that is then interpreted by the cell.

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Modern molecular biology is built upon our understanding of the structure and function of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) and the enzymes and proteins that interact with these structures. In this section of the course the structures of RNA and DNA will be explored along with the processes by which they are used to transmit information.

The structure of DNA (shown on the left below) is the molecule upon which the Central Dogma of modern molecular biology is based. It contains the information necessary to code for the RNA and proteins used by a cell or virus to replicate and produce the next generation. While a virus does not satisfy one of the major tenets of the Cell Theory that the entity is able to self-replicate, it still uses information from either its own DNA (RNA) or its host DNA to replicate itself.

Although RNA has much of the same basic structural features of DNA, it takes on many more tertiary structures and has multiple functions in the cell. The forms of RNA include:

  1. mRNA: messenger RNA is a copy of the DNA sequence that is read by the ribosome during protein synthesis. The mRNA contains the information on the order of the amino acids in protein that is being synthesized on the ribosome. Each base triplet corresponds to one amino acid.
  2. tRNA: transfer RNA is responsible for translation of the nucleic acid code to an amino acid. tRNA carries an amino acid to the protein synthesis machinery (ribosome) and is responsible for decoding the mRNA information to insert the correct amino acid into the growing protein on the ribosome. tRNA folds into a distinct three dimensional structure, as shown in the 3D Jmol image on the right.
  3. rRNA: ribosomal RNA is a significant structural component of the ribosome and plays a role in the catalyzing the formation of the peptide bond during protein synthesis. Since the rRNA catalyzes the reaction, it is referred to as catalytic RNA or a ribozyme.

Combining all of these roles together, the Central Dogma of modern molecular biology follows the process outlined in this section and depicted below. DNA, with the aid of specific proteins and enzymes is replicated (DNA Replication) thus providing a copy to be passed to the next generation, the DNA is transcribed in sections (Transcription) into a RNA molecule that codes for a protein or that can itself be used in the form of RNA, and the RNA can be translated (Translation) into the primary sequence of a protein.

This short animation summarizes the processes central to Molecular Biology. The DNA double strand separates, the RNA Polymerase moves along the strand and transcribes the information from one strand of the DNA to a strand of RNA, and the RNA leaves the nucleus where its sequence is translated to a protein sequence on the ribosome.

The intensive efforts to understand the structure of the molecules involved as well as the details of the process have also yielded a set of tools that have led to the sequencing of whole genomes containing the inherited information passed from generation to generation. This immense amount of information has spawned the field of computational biology that is able to extract information from the sequences that can be applied to both our basic understanding of the functioning of organisms and to applications leading to potential cures for genetically inherited diseases.

The Role of DNA and RNA

This section begins our description of the structure and function of DNA and RNA. The ultimate tertiary structures of RNA and DNA are dependent on both the similarities and differences in the primary structure of each of the polymers.

Both DNA and RNA are linear polymers of building blocks. Each block contains a planer nucleotide base that is joined to a sugar, either a deoxyribose in the case of DNA or a ribose in the case of RNA. Each block is joined by a oxygen-phosphate-oxygen bridge. The alternating ribose-phosphate-ribose is referred to as the backbone of the nucleic acid polymer, in much the same way the N, alpha-Carbon, and carbonyl atoms in an amino acid form the backbone of a protein. Similarly, the nucleotide bases are analogous to the amino acid sidechains.

Protein and Nucleic Acid Structures
The main features of a protein (top) and a nucleic acid (bottom) polymer are illustrated. Both polymers contain repeating units (amino acids, ribo-bases) linked by bonds (peptide, phosphodiester) to give linear polymers. The mainchain atoms form the linear chain. The sidechains project off of the mainatoms, these are either amino acid sidechains (proteins) or bases (nucleic acids). There is also a defined direction to the chain. In the case of proteins the free amino terminus defines the beginning, and the sequence is simply the order of the amino acids, named here by their sidechains (side1-side2...). In the case of nucleic acids, the ribose has a 5' end and a 3' end. The 5' end is considered to be the beginning and the nucleic acid sequence is given as the order of the bases, beginning at the 5' end.

As with proteins, the bases in the nucleic acid can interact with each other to form complex structures. The most important type of interaction between the bases is hydrogen bonding. If bases on two strands have complimentary hydrogen bonding properties they can form a basepair. Double stranded DNA consists of two anti-parallel hydrogen bonded strands. RNA can also exist as an anti-parallel two stranded structure or it can assume more complicated structures, such as the tRNA molecule shown on the previous page. The detailed chemical structure of DNA and RNA will be explored in more detail in the following pages.

The Backbone

Both DNA and RNA are linear polymers. The components of the backbone of the polymer include a set of furanose sugars linked together by bridging phosphate molecules in a synthesis between the 3 position of one furanose and the 5 position of the next furanose. This linkage is made through condensation synthesis formation of ester bonds by a bridging phosphate molecule between two hydroxyl groups, one on each furanose ring. The resulting polymer is a string of furanose molecules linked by phosphodiester bonds and having the 5 position exposed on one end of the polymer and the 3 position exposed on the other end of the molecule.

Polymer

The major difference in the polymer backbones between DNA and RNA is the sugar used in the formation of the polymer. In DNA (DeoxyriboNucleic Acid) the sugar is the aldose deoxyribose with a hydrogen replacing the hydroxyl at the 2 position of ribose, a furanose. In RNA (RiboNuceic Acid), the sugar is the monosaccharide ribose in the furanose conformation.

The numbering of the positions on the sugar furanose rings of DNA and RNA follow a convention that uses ' (prime) to denote the sugar positions. Thus the ribose has X connected to the 1' position and hydroxyl groups on the 2', 3' and 5' positions. Using this nomenclature, deoxyribose is formally called 2'-deoxyribose (2 prime deoxyribose) to denote the loss of the hydroxyl at the 2' position of ribose.

Furanose Sugars

And, the resulting phosphodiester link is between the 3' position of one furanose and the 5' position on the second furanose.

Formation of phosphodiester Bonds
Condensation synthesis of the phosphodiester bond between the 3' position of one ribose and the 5' position of the second ribose.

The images below show the backbone structures for both DNA and RNA.

Phosphodiester linked sugar-phosphate-sugar backbone
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The synthesis of the backbones illustrated above is directed by specific enzymes that restrict the possible structures to links between the 3' position and the 5' position of the sugars with a bridging phosphate and to selected sugars with specific inclusion of either ribose or 2'-deoxyribose.

The following is a list of structural characteristics of the DNA/RNA polymer backbone.

  • Phosphate-ribose(deoxyribose)-phosphate-ribose(deoxyribose)sequence
  • Linked by Phosphodiester covalent bonds
  • 3' position on one ribose(deoxyribose) linked to 5' position of adjacent ribose(deoxyribose) through phosphodiester bridge
  • chain has 3' end and 5' end

In the structure above the X represents the bases that distinguish the units of the backbone from each other in much the same way that the 20 naturally occuring side chains on a common backbone distinguish the units of a polypeptide (protein) structure. These bases will be explored in the following sections.

The following is a video in which Dr. Brown explains the structure and formation of DNA and RNA backbones.

The Bases

The backbone structure of both DNA and RNA shows variability according to the composition of X in the structure given on the previous page. X represents a set of nitrogenous bases. The bases are divided into two fundamental ring structures: purines (2 fused aromatic rings) and pyrimidines (a single aromatic ring). The differences in the bases, which are only found attached to a sugar, give rise to the second major variation in the difference between DNA and RNA.

All of the bases have the common structural characteristic that they are planar structures due to the aromatic (alternating double bonds in the rings) structure of the molecules. This feature parallels that previously seen with the phenyl group [Refer to the Functional Groups simulation in the Glossary to review the structure of the phenyl group]. This aromatic character also gives rise to the ability of the bases to absorb ultraviolet light at 260 nm. This latter feature provides a distinctive means for identifying the presence of DNA and RNA molecules.

Purine
Pyrimidine
2 fused, planar rings one, planar ring

The variations in the structures of the purine and pyrimidine bases are limited to the five that are used during copying of DNA (Replication) or transcribing of DNA into RNA (Transcription). The structures of these nitrogenous bases are given below.

The bases do not occur as free bases in nature but are always bound to a furanose ring. Since the bases are always associated with a sugar in nature, they take on specific names according to their structure. The base by itself has a specific name and the base attached to a sugar (a nucleoside) has a distinct name. The following table gives the names of the purines and pyrimidines as the free base and as the nucleoside with their one letter abbreviation. The naming of the nucleotidesA component of nucleic acids consisting of a sugar, usually ribose or deoxyribose, a purine or pyrimidine base, and a phosphate group attached either at the 5 prime or 3 prime position of the sugar ring. is analogous to the nucleosideA component of nucleic acids consisting of a sugar, usually ribose or deoxyribose, and a purine or pyrimidine base. A compound obtained by hydrolysis of a nucleic acid. with the added specification of the location of the phosphate group. An example of this nomeclature is given below the table with the structures.

Structure of Bases
The Purine and Pyrimidine Bases
Purine Bases Pyrimidine Bases
base nucleoside found in base nucleoside found in
Adenine Adenosine (A) DNA and RNA Thymine Thymidine (T) DNA only
Guanine Guanosine (G) DNA and RNA Uracil Uridine (U) RNA only
. Cytosine Cytidine (C) DNA and RNA
Nucleoside vs Nucleotide
A nucleoside contains only a base attached to a sugar while a nucleotide is composed of a nucleoside to which a phosphate has been added at either the 3' or 5' position.

Linking the nucleotides together, a linear polymer is generated with sequences varying according to the placement of the bases along the final backbone. The following figure shows a tetranucleotide sequence of DNA. The bases, sugars and phosphodiester bonds are alternately highlighted. In the lower left of the frame is a schematic representation of the same sequence.

The Phosphodiester Bonds between the sugars form the backbone structures of the DNA and RNA

The illustration below shows a dinucleotide of DNA and RNA. Note the backbone to which each of the bases is attached. While there are four bases associated with DNA and four with RNA, are they the same set in each case?

Dinucleotides of DNA and RNA
Replacing the X's on the Backbone of DNA and RNA

The following link is a video in which Dr. Brown explains the structure of bases.

Hydrogen Bonding Between Bases

The following is an interactive simulation that allows you to form hydrogen bonding pairs between the appropriate bases in DNA and RNA that would be allowed in the formation of the double helical structure. You should apply the definition of the hydrogen bond to form all possible hydrogen bonds in any pair of bases you choose. All of the possible hyrogen bonds may be useful later as we explore multiple structures especially for RNA. The focus of this exercise is to itentify bonding partners that will be optimal in the formation of the DNA and RNA helical structures.

Select the proper base from the right to bond with the example on the left. Draw the hydrogen bonds between then and then click the Done when you have drawn all of the bonds and then identify the bases.
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Hybridization of DNA and RNA

On the previous page you determined that the most stable complimentary base pairing takes place between A and T with two hydrogen bonds and between G and C with three hydrogen bonds in DNA. Combining that finding with the backbone information that described DNA as containing A, T, G, and C as the possible bases and A, U, G, and C as the possible bases in RNA, the complimentary base pairing in RNA would include A with U (examine the difference in structure between T and U) with two hydrogen bonds and G with C as described in DNA. In each case these comlimentary base pairings included a purine hydrogen bonded to a pyrimidine. This means that the distance between the attachment sites for the sugar in all the base pairs are identical giving uniform dimensions to the distance between the two backbones along the length of two strands that are hydrogen bonded together.

Because T and U are identical in the hydrogen bonding that each makes with A, this means that a backbone (strand) of DNA can hydrogen bond with another strand of DNA but also with a strand of RNA. The ability of the strands of DNA and RNA to hydrogen bond with each other either as homodimers (DNA-DNA, RNA-RNA) or as heterodimers (DNA-RNA) is referred to as hybridization.

As a general rule, hybridization will take place to maximize the number of hydrogen bonds that can be formed between two polynucleotide strands. However, another structural limitation is placed on the formation of the hydrogen bonds between two lengths of polynucleotide strands: the strands must be anti-parallel to each other. This means that the since the backbne has a 5' end and a 3' end, two strands hydridized to each other must have one strand oriented 5' to 3' and the complimentary strand 3' to 5' as illustrated below.

5'- G- C- A- U- A- G- -3'
3'- C- G- U- A- U- C- -5'

This short duplex structure is anti-parallel and has 15 hydrogen bonds holding the two strands together. As written this is a duplex of two strands of RNA. The figure below shows the same structure as a duplex of DNA.

5'- dG- dC- dA- dT- dA- dG- -3'
3'- dC- dG- dT- dA- dT- dC- -5'

Can you generate a hybrid structure using the top 5' to 3' DNA strand from the figure above and create the appropriate RNA strand hybridized to it?

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Stability of DNA - Base Stacking and Hydrogen Bonds

The stability of double stranded DNA is due to two factors, hydrogen bonds and base stacking. Hydrogen bonds provide an attractive force between the strands while base stacking (van der Waals) stabilizes the helical structure. The Jmol below shows the stacking of the bases and illustrates the hydrogen bonding pattern for a GC basepair. The stability of the DNA is to some extent determined by the number of hydrogen bonds holding the two strands together. Thus DNA with more GC base pairs is likely to be more stable than one with more AT base pairs.

Experimentally it is possible to measure this stability by following the melting of DNA with increasing temperature. Below is a graph of the melting of DNA that coincides with an increase in the ultraviolet (UV) absorbance of the DNA at 260 nm. The increase in absorbance is referred to as the hyperchromic effect and is a measure of the breaking of the hydrogen bonds between the bases and the separation of the two strands.

Measurement of Melting Temperature (Tm) of DNA
The melting temperatre (Tm) for a piece of DNA is the temperature at which 50% is no longer hybridized.

The midpoint of the transition from the double stranded to the single stranded form of the DNA is called the melting temperature (Tm) for the DNA. In most organisms the Tm of the chromosomal DNA ranges from 85-100 degrees C. It is possible to determine the composition of the DNA experimentally from its Tm because the Tm of DNA is directly proportional to the GC content of the DNA as graphically illustrated below.

Measuring the composition of DNA
The melting temperature is directly proportional to the composition of the DNA. This is also a measure of the hydrogen bonding content of the DNA.

While the melting temperature does not tell us anything about the proteins that are coded by the DNA, it does tell us something about the tolerance of the organism in which the DNA is found. For example, thermophilic bacteria (those that survive at extremely high temperatures) have DNA with very high Tm values so that their DNA does not melt at their normal environmental temperature. As we will see later, this also has implications for how the same proteins can be made in two different organisms using DNA with vastly different compositions.

In summary, for the hybridization of two strands of DNA and RNA

  1. the chains are antiparallel
  2. the two chains are held together by hydrogen bonding between bases
  3. the stability of the DNA is directly proportional to the number of hydrogen bonds between chains
  4. hydrogen bonding between strands of DNA and RNA follow the pattern
    • A hydrogen bonded through two bonds to T in DNA
    • A hydrogen bonded through two bonds to U in RNA
    • G hydrogen bonded through three bonds to C in both DNA and RNA

Building the Double Helix

As described in the previous section, the hybridization of DNA and RNA results in the formation of:

  • Double stranded DNA or RNA with
  • Antiparallel orientation (5’ to 3’ against 3’ to 5’) with
  • Uniform distance between the strands due to pairing of a purine with a pyrimidine (A with T (or U) and G with C).

This representation of the ladder of a double stranded DNA is illustrated in the figure below on the left side. However, this secondary structure spontaneously forms a double helical coiled structure that is depicted on the right hand side of the figure below. This structure has several distinct features that characterize the dominant structure called B-DNA.

Use the interactive 3D Jmol image to explore these features of the double helix:.

  • The two helical polynucleotide chains are coiled around a common axis
  • The phosphate and ribose groups are on the outside
  • The bases are directed toward the inside of the helix, and stack on the central helical axis.
  • The planes of the bases are perpendicular to the axis of the helix
  • The diameter of the helix is uniform and is 20 Angstroms
  • The bases are stacked and separated by a uniform distance of 3.4 Angstroms.

The following link is a video in which Dr. Brown explains how DNA and RNA are constructed from the backbones and bases.

DNA has Grooves

A predominate feature of the DNA double helix is the presence of grooves, or indentations, on the side of the helix. This grooves expose the edges of the basepairs, The grooves are called the major groove and the minor groove. Both grooves are deep and expose the edges of the bases to the external environment, making them accessible for protein binding. The minor groove is quite narrow (approximately 12 Angstroms across) and while the edges of the bases may be accessible to solvent and small molecules, they are generally not accessible to larger molecules. The major groove on the other hand is quite wide (approximately 22 Angstroms across) and is sufficiently wide to accommodate a protein alpha helix. Below is a side view of the B-DNA structure using a space filled model (left) next to a 3D Jmol image that will allow you to highlight each groove and determine which atoms from each basepair project into the groove. The nature of the atoms that are exposed in the grooves of the structure are important for the ability of proteins to recognize specific sequences of DNA. Discrimination of the different sequences must be made by having access to the bases inside the structure since the backbone structure is common to all sequences of DNA


Side View of B-DNA Helix
This space filled model of the B-DNA shows the major and minor grooves resulting from the formation of the double helix.

DNA Structure (B-helix)

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Bases pairs inside double helix
Use the buttons on the above Jmol to see the location of a C-G and a T-A basepair in the double helix.
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The tertiary structure of B-DNA has been described with a number of specific characteristics. These characteristics describe what is referred to as the B-helix. It is the most common structural form of double stranded DNA. RNA and hybrids of DNA with RNA have altered characteristics resulting from the substitution of a ribose for a deoxyribose in the backbone structure. The general characteristics found in the B-DNA are still present; however, slight differences in the specific characteristics result in dramatic changes in the overall structure of the helix.

The following table compares some of the finer characteristics of the B-helix and A-helix. In general, the A-helix is broader than the B-helix, there are slightly more base pairs per turn in the A-helix, and most significantly, the tilt of the bases relative to the axis of the helix is much greater in the A-Helix.

A-helix B-helix
Shape Broader than B
Screw Sense Right Right
Base pairs/Turn 11 10.4
Pitch/turn 25 35
Tilt of Bases 19 1
Major Groove Narrow/Very Deep Wide/Quite Deep
Minor Groove Very Broad/Shallow Narrow/Deep

The following 3D representations illustrate the result of these differences in comparing similar views of the B-helix of DNA with the A-helix in duplex RNA. When exploring these structures you should focus on the following aspects of the structures, noting similarities and differences:

  1. The location of phosphate, ribose, and bases with respect to the interior and exterior of the helix
  2. The location of the major and minor grooves. The grooves are water and ion filled channels on the side of the double helix. Proteins usually interact with the edges of the bases in the major groove.
  3. The tilt of the bases with respect to the axis of the helix.
  4. How the bases overlap with each other. As a result of the greater tilt in the bases relative to the helix axis, the bases of the A-helix slide past each other and do not exactly stack on top of each other as they do in the B-helix.

As discussed at the beginning of this unit, modern molecular biology has developed a Central Dogma that describes a series of processes starting with DNA and ending with the production of a protein using the genetic code. This Central Dogma is diagrammed in the figure below. The initiation of this process is DNA Replication, highlighted in red, which describes the copying of the information existing in DNA to new DNA. One of the most important processes that a cell performs before it can divide is to faithfully replicate its chromosome. While there are differences between prokaryotes and eukaryotes, there are great similarities in the mechanism by which they replicate their DNA. This unit will describe this mechanism of DNA replication

In addition, an understanding of the enzymes that are involved in faithfully copying the DNA has also lead to their use in applications that have advanced our knowledge of genomes, the information they contain and how they are used. The polymerase chain reaction (PCR) and DNA sequencing using dideoxynucleotides have revolutionized our ability to work with small amounts of DNA and generate immense amounts of sequence information in a very short time. The understanding of the basic principles involved in DNA replication have also lead to our understanding of such topics as DNA repair and chromosome extension.

DNA replication is the process by which DNA is copied resulting in a faithful replicate of the original double stranded DNA. Key to the inheritance of the code for materials produced and used in a cell, DNA replication must be faithful thus not allowing or minimizing errors during the process and it must generate new DNA that can be transmitted to the next generation. Because DNA replication generates a copy of the original, it is referred to as DNA dependent DNA synthesis. It is dependent on DNA because it must have a template to use in making the copy and the result is a new strand of double stranded (duplex) DNA.

Semi-conservative Replication

The process of DNA replication requires that the original DNA act as a template for the newly formed DNA. How the new DNA acts as a template could occur in multiple ways. As depicted below the double stranded DNA (anti-parallel strands indicated by the grey and opposite polarity blue strand) could be completely copied leaving the original strands of DNA intact (grey) and yielding a newly synthesized double strand DNA containing two new strands (blue). This is referred to as conservative replication. The second model of DNA replication starts with the same double stranded DNA but the products each contain one of the original strands and a complementary newly synthesized strand. This is referred to as semi-conservative replication. Biological organisms have been shown to replicate their DNA by semi-conservative synthesis.

The “Learn More…” that follows shows the Meselson-Stahl experiment that was performed to prove that semi-conservative replication is the mechanism within biological systems.

Semi-conservative vs Conservative replication models
Semiconservative vs Conservative Replication

DNA synthesis

DNA synthesis in biological systems is a very directed process controlled by specific enzymes that both dictate where synthesis begins and the exact structural conditions required to undertake DNA synthesis. Knowledge of these conditions and the process involved in DNA synthesis has become the basis for understanding other biological processes involving DNA and in the design of a set of tools essential to the advancement of the study of modern molecular biology.

DNA polymerases (setting the rules)

DNA polymerase is an enzyme that has very specific substrate requirements and thus controls where the synthesis of DNA can take place. An understanding of the basic mechanism of this enzyme will make the understanding of how DNA replication works become clear.

  1. As with any enzyme, DNA polymerase has a very specific set of requirements for recognition of its substrate. For DNA polymerase the substrate is single stranded DNA not double stranded DNA. Thus DNA polymerase is not able to bind to or recognize an intact genome but rather it uses each single strand of DNA as a template.
  2. While DNA polymerase requires single stranded DNA as a template, it does not have the ability to start anywhere on the strand and initiate de novo synthesis. The active site of the polymerase requires a segment of DNA or RNA bound in a complimentary manner to the single stranded template as an initiation point for the synthesis reaction. This segment of DNA or RNA is generally referred to as the primer.
  3. If you examine the structure of either DNA or RNA there are two places that a new deoxy-nucleotide can be added to extend the length of the complementary strand.
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While DNA synthesis could be from either end of the primer, during replication using the enzyme DNA polymerase the synthesis is restricted by the enzyme to growth in only one direction. Deoxy-nucleotides are only added to the 3’ end of the primer and thus synthesis is unidirection from 5’ to 3’.

  1. For synthesis to proceed, the enzyme imposes a third restriction on the substrate. Not only does it require a DNA template and a primer, but the primer must have a free, 3’ hydroxyl group for the addition of the next deoxy-nucleotide.
  2. And finally, all four deoxy-nucleotide triphosphates must be present to completely synthesis the complementary strand of DNA. For DNA synthesis (replication) this means dATP, dTTP, dCTP, and dGTP must be present. Collectively these are referred to as dNTPs (deoxy-Nucleotide TriPhosphates).

More than one DNA polymerase enzyme is known and each carries out a specific function depending on the situation. For example, DNA polymerase III is the enzyme used in replicating a genome while DNA polymerase I is used in DNA repair. Even with clear differences as to when each of the DNA polymerases is used during the replication of DNA, the basic substrate requirements are the same.

The following diagram depicts the structural requirements for the operation of DNA polymerase enzymes.

Perfect Substrate for DNA Polymerase
Perfect substrate for DNA Polymerase

Synthesis (the making of new DNA)

Once the appropriate structural requirements for the synthesis of DNA are met, DNA synthesis occurs continuously in a unidirection process from 5’ to 3’ along the template. The following animation demonstrates the building of a complementary strand using the components required by DNA polymerase. This process can take place wherever the initial conditions for operation of DNA polymerase exist.

DNA Polymerase binds to DNA at the 3' end of the primer and builds a complementary sequence.

Initiation of synthesis from double stranded DNA

Having explored the requirements for DNA polymerase, it is clear that the prokaryotic and eukaryotic genomes fail the first requirement of a single stranded sequence of DNA. Therefore to initiate DNA replication it is necessary to create the conditions that will allow DNA polymerase to carry out DNA synthesis.

  1. First the DNA must be opened by breaking the hydrogen bonds between the base pairs and unwinding the DNA. This is initiated by a protein called the initiator protein or DnaA that recognizes a specific DNA sequence called the origin of replication, binds to the sequence and opens up the sequence to create a replication bubble (open complex). An enzyme called a helicase binds to the open complex and extends the melted region of the double stranded DNA to further open the replication bubble. There now exists a single stranded segment of DNA exposed on both chains of the DNA double helix. A small protein called SSB (Single Stranded Binding protein) coats the open complex.

The Open Complex

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  1. With formation of an open complex, one of the requirements for DNA polymerase is satisfied: single stranded DNA is generated. At either end of the open complex where the single stranded and double stranded DNA meet, a structure referred to as the replication fork exists. From this point we will focus on the process of replication at one of the replication forks. Remember that the replication will be occurring simultaneously from both forks in opposite directions. To allow the binding of DNA polymerase, a second requirement must be met for the structure of the substrate. An enzyme called Primase synthesizes a short (approximately 10 nucleotides), complementary RNA primer on each strand of the DNA in a 5’ to 3’ unidirectional fashion. This complex of DNA and protein at the replication fork is referred to as the replisome.
Replisome at one fork
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  1. The substrate requirements for DNA polymerase have been satisfied and synthesis begins. Helicase continues to melt the DNA in front of DNA polymerase.
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  1. As single stranded DNA is generated between the ‘back’ of the RNA primer on one strand and the opening replication fork, a new segment of RNA primer is laid down near the opening fork and DNA polymerase synthesizes a new complementary strand of DNA from the primer toward 5’ end of the existing primer and stops. This segment of DNA that is not connected to the next segment is referred to as an Okazaki Fragment. Synthesis on this lagging strand is said to be discontinuous since it is generated in uniform lengths of DNA starting from one primer and stopping before the next RNA primer.
  2. Synthesis on the opposite strand is a continuous uninterrupted process starting from the initial primer and continuing as long as the DNA continues to open up at the replication fork. This strand on which continuous synthesis takes place is referred to as the Leading Strand.
Okazaki fragments on the lagging strand

The following animation depicts the complete process of DNA replication. In the animation you should be able to identify each of the stages of synthesis on each of the strands. At the end of the synthesis you will see that the leading strand has a continuous double helix generated while the lagging strand has a discontinuous set of Okazaki fragments that must be connected before the synthesis is complete.

To accomplish this mending of the DNA, a separate DNA polymerase binds to the ‘nick’ left in the DNA between the 3’ end of the newly synthesized DNA and the 5’ end of the RNA primer. The substrate requirements for DNA polymerase are met even though there is no apparent single stranded DNA present. The DNA polymerase removes the RNA primer and a few nucleotides of DNA in front of it while simultaneously synthesizing DNA on the 3’ end of the existing DNA. This process is referred to as nick translation.

When only DNA is present, the DNA polymerase releases from the DNA leaving a nick in the DNA between two DNA segments. DNA polymerase does not have a function to join the two ends of the DNA together and thus a separate enzyme, DNA Ligase, recognizes the nick in the DNA and links the 3’ end of the one segment to the 5’ end of the next segment.

This process of DNA replication continues until the entire sequence of DNA is synthesized. The process is fundamentally the same in prokaryotes and eukaryotes.

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Solving Challenges within the Rules

The Origin of Replication – where to start

The Challenge: To initiate the DNA replication of a chromosome the appropriate substrate conditions must be met as described previously. How long the process of replication takes is also important to the cell. How genomes of variable sizes can be totally replicated within the appropriate life cycle time of the cell is a significant challenge.

Bacterial chromosomes have a single point of origin for replication

The origin of replication has been described as a specific DNA sequence to which initiator protein (DnaA) binds and initiates the process of creating the open complex for synthesis. For a typical prokaryotic (bacterial) chromosome there is one origin of replication and synthesis proceeds at a rate of approximately 500 bp(base pairs)/sec. A bp or base pair is the unit of length of DNA measured in single nucleotides or hydrogen bonded base pairs along the polymer chain. Thus if a typical bacterial chromosome is 2x10e6 (two million) bp in length then the replication time of the chromosome would be about 30 minutes which is consistent with the life cycle of the typical bacterial being 30 to 40 minutes.

Eukaryotic chromosomes have multiple points of origin for replication

The replication rate for the eukaryotic replication fork has been measured to be approximately 50 bp/sec. This means that for a eukaryotic chromosome of 10e8 bp to completely replicate starting at one origin of replication it would take approximately 23 days. While we know that the life-cycles of eukaryotic cells are extremely variable, the S-phase, the period of time during the eukaryotic life-cycle when DNA is replicated, typically last 8 hours. To explain the enormous difference in time scale, it has been found that the eukaryotic chromosome has multiple origins of replication. The eukaryotic replication units have upwards of 80 origins which means the replication time is reduced to approximately 7 hrs.

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What if DNA polymerase makes a mistake

The Challenge: DNA polymerase synthesizes DNA at rates up to 500 bp/sec. There are basically three types of mistakes that the enzyme can make during the synthesis: substitution of an incorrect base, insertion of an extra base or failure to insert a base resulting in a deletion. The consequences of these errors will be apparent as we move toward the discussion of DNA transcription and RNA Translation. In the context of Replication it is important to touch on at least a couple of the means by which the system corrects errors or reduces the frequency of errors.

Editing the Synthesis

DNA polymerases are members of a very interesting family of enzymes because of the multi functional nature of their activity. As has been discussed previously, they are capable of synthesizing DNA given the appropriate substrate. They have their own activity to remove RNA and/or DNA in front of the synthesis as described on the lagging strand of the replication fork. Equally important to the organism is the ability to edit errors during synthesis by either preventing the incorporation of the incorrect base and/or by removing an incorrectly incorporated base before the next base is added. These editing functions can reduce the error frequency by 2 orders of magnitude.

However, even this editing function does not get the error rate to an acceptable number for survival of an organism. In addition to editing during synthesis, there is also monitoring of the DNA for missed errors resulting from replication and for post-replication damage to the DNA. In each of these cases the process involves cutting one of the strands of the DNA specifically creating a condition by which a DNA polymerase can replace the error or damaged DNA followed by ligation of the nicked DNA using DNA ligase. It is to be noted that while different proteins may be used, the basic process is the same and follows the direction/rules dictated by the mechanism of the specific enzymes. The combination of these other editing and repair functions reduces the error rate by another 2 orders of magnitude thus reducing the error frequency to an acceptable level.

Following the rules

Understanding the process/mechanism of DNA replication has allowed researchers to understand how certain DNA repair functions are conducted and to develop new tools that have revolutionized our ability to gain information from DNA.

Overcoming Chromosome Shortening

During replication of circular chromosomes, such as in bacteria, the leading strand of synthesis from one replication fork eventually meets the lagging strand of synthesis for the other replication fork and there is complete synthesis of the entire chromosome. For linear chromosomes this same process happens between two origins of replication. However at the end of the linear chromosomes, the telomeres, the lagging strand does not completely replicate to match the length of DNA generated on the leading strand. Thus for repeated replication of the chromosomes during multiple cell divisions, this means that the chromosomes are shortened with each cycle of replication.

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Since DNA synthesis is always unidirectional from 5’ to 3’, it is necessary to lengthen the 3’ end of the chromosome such that primer directed synthesis can take place to lengthen the shorter 5’ end of the chromosome. While the staggered end of the chromosome is not avoided, the net length of the chromosome is recovered or even lengthened. The diagrams below demonstrate how lengthening of the chromosome can be accomplished using the enzyme telomerase. The telomerase enzyme is a polymerase like enzyme that carries its own template with the template having overlap complementarity with the 3’ end of the chromosome. The criteria for polymerization are met, a template and a primer with a free 3’ hydroxyl, and the telomerase lengthens the 3’ strand of the chromosome. This process of lengthening is repeated until a the normal replication process lays down a RNA primer using the primase enzyme, DNA polymerase synthesizes new DNA from the 3’ end of the RNA primer and DNA ligase joins the newly synthesized DNA to the 5’ end of the chromosome.

Process of Telomerase lengthening the 3’ strand of the chromosome.

Being creative with the rules

Two tools that have become integral parts of the study of modern biology were developed through the simple understanding of how the DNA replication process works. At the beginning of this unit three structural requirements for the substrate of DNA polymerase were described. Satisfying those requirements is the basis for the polymerase chain reaction (PCR), used to amplify segments of DNA, and the Sanger DNA sequencing method used in sequencing the human genome as well as others.

Polymerase Chain Reaction

Characterization of large numbers of microorganisms, rapid screening for genetic diseases and identification of individuals or species from very small amounts of tissues or cells requires a means to amplify a specific sequence of DNA for further manipulation. Previously this would have required growing large quantities of cells containing the DNA or obtaining large quantities of the tissue. DNA replication is the process of doubling the original amount of DNA. The polymerase chain reaction (PCR) is simply a method by which DNA replication is repeated through many cycles. In the same way that doubling a bacterial cell through 20 cycles will generate a million copies of the bacteria, replication of DNA through 20 cycles will amplify the amount of DNA by a million fold.

View this PCR tutorial for a quick overview of the process. (©Sumanas, Inc.)

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PCR is limited by the length of DNA that can be repeatedly replicated by DNA polymerase. Different polymerases and different conditions are described to amplify DNA of varying lengths. Routinely, segments of DNA from 500 to 1000 bp in length are easily replicated. The segment of DNA to be replicated is defined by the position of the primers that define the start of the replication on each strand. These are referred to as the forward primer and the reverse primer. The double stranded DNA to be amplified is melted by raising the temperature high enough to fully melt the DNA creating single stranded DNA. The sample is cooled in the presence of the primers and each primer binds to its complementary strand. The appropriate substrate conditions for DNA polymerase binding and synthesis are now in place. DNA polymerase is added with the dNTPs and synthesis of DNA proceeds for as long as the reaction is allowed to progress before either the time of the reaction ends or the DNA polymerase falls off the substrate.

The temperature is raised again to melt the DNA and create a single stranded template.

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Because DNA polymerase will generally denature and irreversibly inactivate at these elevated temperatures it is necessary to add fresh DNA polymerase at each cycle when the temperature is lowered during primer attachment. To avoid repetitive addition of a very expensive enzyme after each cycle and to make automation of the process feasible, there was an advantage to finding a DNA polymerase that survives high temperatures and in fact is active at high temperatures. Such an enzyme was found from thermophilic bacteria (bacteria that thrive at extremely high temperatures). One such enzyme, taq DNA polymerase from thermos aquaticus, is optimally active around 75 degrees and can withstand temperatures as high as 100 degrees without denaturing. Thus the PCR reaction can be performed in a closed tube with DNA to be amplified, taq DNA polymerase, excess dNTPs, and excess amounts of the forward and reverse primers. The reaction tube is put in an instrument called a thermocycler that can be programmed to automatically raise and lower the reaction temperature. Now the temperature is raised to form single stranded DNA, lowered to bind the primers and DNA polymerase with the correct substrate again replicates the DNA. After 20 cycles of this process with each cycle doubling the number of duplex DNA strands present, there will be 1, 048,576 copies of the DNA present.

DNA Sequencing

Current methods of DNA sequencing rely on understanding the substrate requirements of DNA polymerase and the concept of competition. To sequence DNA as with PCR it is necessary to create single stranded DNA that will act as the template. It is generally required that to ensure valid DNA sequence, a double stranded segment of DNA should be sequenced in both directions using primers at each of sequence to be defined. These primers are the equivalent of the forward and reverse primers of PCR. However, where PCR replicates both strands simultaneously, DNA sequencing is performed on each strand individually. Starting with the single stranded template and the bound primer with a free 3’ hydroxyl, the conditions are set for DNA polymerase to replicate the DNA.

If all four dNTPs were added at this point simple replication of the strand of DNA would occur and no information would be gained. However, if one of the dNTPs was substituted by a NTP that had no hydroxyl on either the 2’ or the 3’ position, a dideoxyNTP (ddNTP), it would be incorporated into the growing DNA strand through the 5’ position in a complementary position opposite its complementary base on the template strand but synthesis from that point forward would be terminated due to the absence of a hydroxyl on the 3’ position. The requirement for synthesis by DNA polymerase has been lost. The dideoxyNTP is referred to as a suicide substrate.

If complete replacement of dATP with ddATP were made, then the first time a T were encountered in the template strand, DNA polymerase would incorporate ddATP and all synthesis would halt. If however, a mixture of dATP and ddATP were present, then when the first T were encountered in the template, the dATP and ddATP would compete for the active site of DNA polymerase. If dATP is incorporated, synthesis continues to the next nucleotide and if ddTAP is incorporated synthesis halts for that one strand of DNA. Thus in a population of template strands, a fragment of DNA starting at the primer and ending at each T would be generated with the length of the fragment in nucleotide units defines the location of the T in the template strand. If this experiment is performed for each individual nucleotide, the complete sequence of the template can be accomplished.

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The second phase of the Central Dogma is the process of transcription, which is summarized as DNA directed RNA synthesis. This unit will describe the process of DNA transcription and the different categories of products and how each is processed. The discussion will be organized around:

Transcription

The process of DNA transcription is described as DNA-directed RNA synthesis. This process, which takes place in the nucleus of eukaryotic cells, has many similarities with DNA replication. The enzyme to catalyze the process is RNA Polymerase that likewise has some characteristics similar to those of DNA polymerase used in replication but it also has significant differences.

The Operon

The organization of the genetic information on the chromosomes of prokaryotes and eukaryotes is different. In each case, as indicated below, the sequence for initiation of transcription is the Promoter and the sequence signaling the end of transcription is the Terminator. However, in eukaryotes there is generally one gene that codes for one product under the control of a single promoter. In contrast, prokaryotes generally have multiple genes, each coding for a separate product, under the control of a single promoter. This unit of a promoter, a terminator and the intervening gene or genes is called an Operon. The operon also contains the controlling elements for the operon. The control of expression of an operon is the topic for a separate unit.

Eukaryotic Operon Prokaryotic Operon

The Promoter

RNA polymerase is an example of a quaternary structure composed of a core protein for the synthesis the complementary RNA strand and a subunit, the Sigma subunit, that first binds to the promoter region of the Operon and creates an open complex by unwinding the double stranded DNA. The promoter region of the operon is critical in defining how much or the frequency with which transcription of an operon takes place. Promoters have different sequences. The sigma subunit binds to the different promoter sequences with different affinities. In the illustration below, the sigma subunit binds to both of the promoters and a dissociation constant, Kd, can be written for each binding.

Different promoters result in different Kd values.
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Promoters can be characterized as being strong or weak promoters depending on their affinity for RNA polymerase. The result of a strong promoter with a high affinity (tight binding) to RNA polymerase is a greater frequency of starting transcription from that promoter. Therefore, more product from transcription initiated at a strong promoter is possible compared to product formation initiated from a weak promoter in the same amount of time.

RNA Synthesis

Once RNA polymerase binds to the promoter, initiation of RNA synthesis or Transcription proceeds. The following animation depicts the steps of transcription.

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The Products of Transcription

The product of the process of transcription is RNA. There are three distinctly different RNA products that result from transcription based on their function within the cell.

Each of the RNA classes (rRNA, mRNA, and tRNA) is produced during transcription using a different RNA polymerase.

Bioselectivity describes the discrimination of a specific RNA polymerase for a specific promoter yielding different RNA products. Equilibrium binding of the polymerase with the promoter explains the differential production of products using the same polymerase and different promoter sequences.

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Post-Transcriptional Modification

The products of transcription, rRNA, tRNA and mRNA, are each used for different functions within the cell but are all essential to protein synthesis. They each also undergo change or modification before they are used to carry out their specific function. This process of change after transcription from DNA to RNA is called post-transcriptional modification.

For ribosomal-RNA (rRNA)the modification involves cutting of the original long segment of RNA transcript into fragments. The processing of the RNA takes place in the nucleus and the fragments are used in the nucleoli where ribosomes are assembled.

For transfer-RNA (tRNA), the RNA transcript has a segment of the 5’ end removed, a trinucleotide that is common to all tRNAs (ACC) added to the 3’ end, and a short segment of RNA spliced out of the middle of the RNA sequence. In addition, several of the bases in the structure are chemically modified after the final sequence is generated.

The most extensively modified transcript is messenger-RNA (mRNA) in eukaryotic organisms. For this reason, the primary transcript or mRNA before it is modified is referred to as pre-mRNA.

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Recently, research has found that a number of mRNA sequences undergo what is referred to as alternative-splicing. If normal splicing is from the 3’ end of one exon and the 5’ end of the immediately following exon, imagine the number of possible splices that could occur if the 3’ end of the exon could be joined to the 5’ end of any exon downstream from the first exon. For example, if an mRNA had three exons, 1, 2, and 3 in order, and two introns, the normal sequence of splicing would link exon 1 to exon 2 to exon 3. But it would be possible to link exon 1 to exon 3 to create a “new” protein with only two of the three exons. This allows a single gene with multiple exons to represent many different proteins. Errors in splicing have also been found to be the cause of certain diseases.

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The final stage of the Central Dogma is translation, which is defined as RNA Directed Protein Synthesis. This process is unique in that it takes information from one type of polymer (RNA) and translates that linear information into the linear sequence of a totally different polymer, a protein. This unit will describe that process and the elements that are required to have it function. The discussion will be organized around,

The Code

Translation involves the conversion of a sequence of RNA to a corresponding sequence of amino acids. To perform the conversion a code is needed to translate from the four nucleotides (AUGC) of mRNA to the 20 naturally occurring amino acids. The question then is what combination of nucleotides can code for at least twenty different amino acids? Four nucleotides taken two at a time would generate 16 possible unique sequences. Not enough to code for 20 different amino acids. However, four nucleotides taken 3 at a time would generate 64 different unique sequences. Clearly this is enough to code for the 20 naturally-occurring amino acids. In fact, the abundance of codes means that there is redundancy or degeneracy in the code with more than one triplet code representing a single amino acid.

Through experimentation it was found that the universal start code or codon is AUG and three stop codons (UAG, UGA and UAA) were identified. The remaining 60 codons represented the 20 amino acids. Having elucidated the entire code it was found that for many of the amino acids, the first two nucleotides in the sequence defined the amino acid with the third nucleotide being any of the four nucleotides. For example the codon for the amino acid leucine is CUX where the X represents any one of the four nucleotides, and the codon for the amino acid valine is GUX. This third base then became known as the wobble base signifying the flexibility the system has in identifying the third base. This also demonstrates the fact that a single amino acid can be coded by several triplet codes but a triplet code only represents a single amino acid. This triplet code for converting the sequence of mRNA to a sequence of amino acids is referred to as the Genetic Code.

The Starting Materials

Having identified a code to perform the translation, it is now necessary to describe the process by which the genetic code is used. Four major ingredients are necessary to carry out translation: m-RNA, t-RNA, the ribosome and the initiation factors.

Messenger RNA (mRNA) was discussed in the last section with a description of the post-transcriptional modification that convert the pre-mRNA in the nucleus to mature-mRNA in the cytoplasm.

Transfer-RNA (tRNA) was introduced in the section on the structure of DNA and RNA and its production and post-transcriptional modification were briefly described in the previous section. As seen in the illustration below, tRNA is produced as a primary transcript in a linear primary structure. Following processing the tRNA folds into the cloverleaf secondary structure. In this structure it is easy to see the 3’ end of the structure with its ACC sequence that is common to all tRNAs. In addition the bottom loop in the structure (the anticodon loop) contains the sequence that is complementary and antiparallel to a sequence on the mRNA. This secondary structure folds to a tertiary structure in the form of an inverted L. The 3’ end is at one end of the structure and is the site that will carry a specific amino acid that corresponds to the sequence of nucleotides in the anticodon at the other end of the molecule. The process by which the correct amino acid is added to the correct tRNA is referred to as the charging of the tRNA. The formation of the charged tRNA is a two-step process as depicted in the "Charging" animation below.

The Following Learn by Doing contains an animation describing the tRNA structures.

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50S and 30S subunits 70 S Initiation Complex

Protein Synthesis

The following animation describes the formation of the initiation complex, the elongation of the peptide during synthesis and the termination of the synthesis at the termination or stop codon. The process is fundamentally the same for prokaryotes and eukaryotes. The one notable exception is in the binding of the mRNA with the small subunit of the ribosome during formation of the initiation complex. Prokaryotes do not have a 5’cap to identify the end of the mRNA but they do have a consensus sequence called the Shine-Dalgarno-Sequence at the 5’ end of the mRNA. This sequence is used to align the mRNA with the appropriate site on the ribosomal subunit. In eukaryotic organisms the 5’cap provides the alignment along with multiple initiation factors. Following formation of the initiation complex, the synthesis of protein follows the same sequence in both organisms.

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The Consequence or Error

During both replication and transcription errors can be made in the incorporation of the correct bases during the complementary copying of the DNA. In a previous section a discussion of the methods used by the system to minimize these errors has been presented. However, even with editing functions, some errors are generated and result in changing of the reading code used during translation to a protein sequence. These changes are referred to as mutations and can be categorized into four different categories described by the end result of the mutation or change.

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Living organisms consume food, in the form of carbohydrates, fats, and amino acids, to live. The process of metabolism breaks these complex biomolecules into simple molecules with the release of energy. The most common form of energy is adenosine triphosphate (ATP). This high energy phosphorylated compound is used as a energy source in many cellular processes and for bio-mechanical functions, such as muscle contraction or ion transport across membranes. The resultant compounds and energy released from food are used to synthesize complex molecules for the specialized needs of the cell or organism.

The transformation of ingested food to energy and simple compounds, or the synthesis of complex molecules, is performed by a series of enzymatic reactions. Collectively, these enzymes, and their substrates and products, are referred to as a metabolic pathway.

Degradative or catabolic pathways generally release energy and electrons from oxidative processes, i.e.

Metabolism of Glucose

the electrons, which are carried on organic electron carriers, can be used to generate additional energy, or can be used for synthetic purposes.

Synthetic, or anabolic consume energy and are generally reductive, requiring electrons.

In this section of the course we will investigate in some detail the production of energy from glucose and how this process is regulated to maintain homeostasis. Since the entire process of metabolism is complex, it is useful to discuss the general features of the metabolic pathways that are involved in converting sugars, amino acids, and fats to energy. Important features of pathways include:

  1. input and output compounds,
  2. the cellular location of the pathway,
  3. the type of energy the pathway produces.

The location and connections between these degradative pathways is shown below:

OVERVIEW OF METABOLISM. Click the radio buttons to outline in yellow the four key pathways: glycolysis, the TCA cycle, electron transport, and ATP synthesis and show the flow of carbon atoms, electrons, and protons through each pathway. Glucose that is brought into the cell via the glucose transporter can suffer two fates, oxidation or storage as glycogen. Oxidation occurs in glycolysis and the TCA cycle, releasing the carbon atoms in glucose as CO2. Note that oxygen is not used until the end of the electron transport chain. High energy electrons, symbolized as orange balls are carried on organic electron carriers to the electron transport chain. As these electrons move through the four complexes, protons are pumped from the mitochondrial matrix across the inner mitochondrial membrane. As these protons flow back through the membrane via ATP synthase, ATP is generated.

Many of these pathways, with minor modifications, are reversed for synthetic purposes. For example, glucose can be synthesized from pyruvate, fatty acids from acetyl-CoA, and amino acids from intermediates in the TCA cycle.

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Degradative, or catabolic, pathways generally produce energy. They usually begin with a number of different compounds, each of which represents a branch at the beginning of the pathway. These branches meet at a common intermediate, and the remaining section of the pathway is usually a linear segment. In this way a number of complex compounds are converted to a common intermediate, reducing the number of unique steps in the degradation of complex molecules.

Synthetic, or anabolic, pathways generally consume energy. They usually consist of an initial linear segment, followed by branching to complex compounds at the end of the pathway. This strategy allows the use of common simple starting materials for the synthesis of a number of complex molecules.

Common features of all metabolic pathways are:

  1. They contain multiple intermediates (e.g. compounds A, B, C, ....), with small molecular differences between the intermediates.
  2. Each step, or conversion between intermediates, is catalyzed by an enzyme (e.g. E1).
  3. The pathway is regulated to optimize the use of resources.

It is possible to reverse the direction of a metabolic pathways, depending on the needs of the organisms; a degradative pathway can become a synthetic one. Many of the enzymes that catalyze reactions in one direction can be easily reversed, and thus function in both pathways. A small number of steps utilize different enzymes in the forward versus the reverse direction. These enzymes are regulated in a coordinated fashion such that a pathway operates in only direction at time.

Pathways can be:

A is the substrate for the first enzyme (E1) in the pathway, B is the product of the enzyme. The final product of the pathways is compound D. Note that this pathway can be reversed, using compound D to eventually synthesize compound A.
An example of a branched pathway. The direction of the arrows indicate that this pathways is an anabolic, or synthetic, pathway where complex biomolecules D and F are synthesized using the simpler molecule A as starting material. In the reverse direction, the complex molecules D and F would be converted to A, releasing energy.
In this circular pathway, compound A is transformed to compound B by the enzyme 1. A series of transformations eventually convert B back to A, restarting the cycle.
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It is essential that biochemical pathways are regulated, otherwise the cell would waste resources. Some general properties associated with the regulation of metabolic pathways are listed below

There are five general methods by which the flux through a step in the pathway can be regulated. These methods differ in how rapidly they can respond to changes in the environment. Each of these methods is discussed below, with the more rapid form of regulation at the top of the list.

Question: How do product (competitive) inhibitors actually inhibit the reaction of the substrate with its enzyme? Complete the following Activity and then enter you answer on the My Response box below it.

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Allosteric Binding
(Definition)
Allosteric binding causes conformational changes in an enzyme that can either inhibit or activate the enzyme.
Question: How do allosteric regulators differ from feedback inhibitors? Use the following activity to explore this difference.
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Oxidation-reduction reactions, or Redox reactions are common in metabolic pathways. Generally, degradative (catabolic) pathways cause the net oxidation of compounds, releasing energy. In contrast, synthetic (anabolilc) pathways, are generally reductive pathways.

Oxidations involve the loss of electrons.

Reductions involve the gain of electrons.

Here are two mnemonics to help you remember where the electrons go during redox reactions:

  1. LEO GER: "leo [the lion] goes grr". Lose electrons oxidation, gain electron reduction.
  2. OIL RIG: Oxidation involves loss, reduction involves gain.

An example of an oxidation is the conversion of iron from its metallic state, Fe0, to its rusted form, Fe+3, by the loss of three electrons.

Oxidation and reduction of iron. Metallic iron, Fe0, becomes oxidized to Fe+3 (otherwise known as rust) by the removal of 3 electrons.

The above reaction is an incomplete description of a redox reaction because it does not indicate the fate of the electrons that were obtained from iron. Since free electrons generally cannot exist, all oxidation reactions must be coupled to a corresponding reduction. Since the above reaction only describes one-half of the reaction it is referred to as a half-reaction. The oxidation of iron could be coupled to the reduction of copper ions, which is described by the following half-reaction:

Reduction of copper ion from its +2 state to +1 state by gain of an electron.

The complete reaction, balanced such that there are no free electrons, is:

A balanced redox equation. Three copper2+ ions provide a total of three electrons to oxidize metallic iron to Fe3+. Note that there are no free electrons.

The pair of compounds that exchange electrons are often referred to as a redox couple.

Redox Carriers

In most biochemical redox reactions a total of two electrons are transferred. These electrons are often transferred as hydrogen atoms, containing a proton and electron. Two common electron acceptors are NAD+ (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). They both can accept two electrons, giving the reduced forms NADH and FADH2, respectively. The structure of the oxidized forms of these compounds are shown below.

The chemical structures of nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD) are shown. These two compounds are commonly used as electron acceptors in metabolic pathways. The portion of each molecule that accepts electrons during the reduction process is highlighted in yellow.

Oxidation of NAD+. In the oxidation of glyceraldehyde to phosphoglycerate an aldehyde is oxidized to a carboxylic acid and the released electrons are placed on to NAD+ to form NADH.

The oxidation of an aldehyde to a carboxylic acid. The two electrons released by the aldehyde are transferred to NAD+ to make NADH. In this diagram only the portion of NAD+/NADH that undergoes chemical changes is shown. The remaining part of the NAD molecule is represented by 'R'.

Oxidation of FAD. The oxidation of succinate to fumarate, using FAD as an electron acceptor is another example of a redox reaction found in a metabolic pathway. Two hydrogen atoms (= two electron plus two protons) are removed from succinate and placed on FAD, producing fumarate and FADH2, oxidizing a carbon-carbon single bond to a double bond.

The oxidation of an alkane to an alkene. The two electrons released by the alkane are transferred to FAD to make FADH2. In this diagram only the portion of FAD/FADH2 that undergoes chemical changes is shown. The remaining part of the FAD molecule is represented by 'R'.

Balancing Redox Reactions

It is often difficult to determine from the structure of an organic compound whether it has been oxidized or reduced in a reaction. For example, the addition of a water molecule to an double bond (alkene) appears to be a redox reaction because an -OH group has been added .

The addition of water to a double bond is a common reaction in many pathways. Is it a redox reaction?

The rules for balancing redox reactions are as follows:

  1. Make the number of oxygen atoms in the reactant and product equal by adding the appropriate number of water molecules to one side of the reaction or the other.
  2. Use H+, or H+ + e-, or e- to balance hydrogen atoms and/or charge.
  3. A redox reaction has occurred if electrons are consumed or released

The above reaction is balanced as is, and is therefore not a redox reaction.

Try the following mini-tutor to test your skill at assessing whether a redox reaction has occurred.

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The operation of a metabolic pathway produces (catabolic) or consumes (anabolic) energy. There are a number of different forms of energy storage that are found in metabolic pathways. These include:

  1. Phosphorylated Compounds Nucleoside triphosphates, such as adenosine triphosphate (ATP) are commonly used to store energy. The addition of an inorganic phosphate group to a nucleotide diphosphate to form the triphosphate requires approximately 30 kJ/mol of energy. The reaction showing the synthesis of ATP from ADP and phosphate is pictured below. Phosphorylation of adenosine diphosphate (ADP) produces adenosine triphosphate (ATP). This reaction requires the input of approximately 30 kJ/mol. Formation of ATP occurs when the negatively charged phosphate group on ADP attacks the electropositive phosphate in inorganic phosphate, forming a phosphate anhydride bond with the release of water (red arrow). ATP can be used to phosphorylate other nucleoside diphosphates with essentially no input of energy, for example:
    The myth of high-energy phosphate bonds. When a phosphate is released from ATP to form ADP, about 30 kJ/mol of energy is released. It is often stated, incorrectly, that the bond that is broken is "high-energy". In fact, its energy is no different than any other phosphate bond of the same type. The release of energy is due to the fact that the products, ADP and inorganic phosphate, are lower in energy than ATP by 30 kJ/mol. One reason that ATP is higher in energy is due to charge repulsion between the negatively charged phosphate groups. Once the phosphate group is removed, the unfavorable repulsion disappears.
  2. Reduced redox carriers.The oxidation of metabolytes usually produces energy, If this energy was not captured in some way, it would be lost as heat. The reduced form of redox carriers, such as NADH and FADH2, are higher in energy than their corresponding oxidized forms, capturing the energy that would otherwise be lost as heat. For example, the oxidation of isocitrate to ketoglutarate releases approximately 70 kJ/mol, 60 of which is captured by converting NAD+ to NADH.
  3. High energy thioesters are often produced by oxidative steps. For example, the energy released by the oxidation of an aldehyde is stored in both the reduced form of NAD+ as well in a thioester. The hydrolysis of the thioester can be used to synthesize nucleoside triphosphates or to facilitate the formation of carbon-carbon bonds, as shown below. The oxidation of the aldehyde to the thioester is highlighted in green. CoA is coenzyme A, a nucleotide containing cofactor that is an essential co-substrate for many reactions. Part of the energy of this oxidation, 60 kJ/mol, is captured by the formation of NADH. The energy stored in the thioester can be used to either phosphorylate GDP to form GTP, capturing another 30 kJ/mol. In the case of acetyl-CoA (lower diagram) the thioester facilitates the attachment of the acetyl group to oxaloacetate to form citrate, the first compound in the TCA cycle.
  4. Proton gradient The transfer of electrons from NADH and FADH2 to oxygen to form water during the electron transport chain, provides energy for the pumping of protons across the inner mitochondrial membrane. This is equivalent to pumping water up hill to fill a reservoir. As the protons flow back through the membrane, the energy released is used to generate ATP, in much the same way water generates electricity in a hydroelectric plant.
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Free Energy and Spontaneity

Pathways accomplish the net conversion of the starting compounds to the final product of the pathway. During the normal operation of a pathway there is a constant flux of material through the pathway in one direction. Note that many pathways are reversible and operate in the forward or reverse direction, depending on the needs of the organism. The direction of a pathway depends on the energy difference between the starting compounds and final product of the pathway. The pathway will be spontaneous in the direction that causes a decrease in the free energy of the system. Reversing the direction of a pathway requires changing the relative energies of the reactants and products

It is useful to have a quantitative way to predict the direction of a pathway given the current environment in the cell. A method of predicting the direction of a reaction was devised by J. Willard Gibbs in 1876 and the quantitative parameter that can be used to predict the direction of a reaction is called the Gibbs free energy. This method is particularly useful because it can be applied to reactions that are not at equilibrium, which is the situation encountered during metabolism. Before we can discuss the Gibbs free energy, we have to discuss standard energies and their relationship to equilibrium positions of reactions. Keep in mind that these discussions relate to the thermodynamic properties of pathways, more specifically the relative energy differences between reactants and products under cellular conditions. The presence of enzymes simply increases the rate of conversion from reactants to products, the enzyme cannot alter the relative energies of these compounds.

Equilibria

Consider the simple reaction [A] to [B]. If we start with a system that is pure [A] it will spontaneously form some [B] until equilibrium is reached. When a system is at equilibrium, the concentration of products and reactants are constant and it is possible to write an equilibrium constant for the reaction:

where, KEQ is:

KEQ=[B]EQ[A]EQ

Note that [A] and [B] are at their equilibrium concentrations in this formula.

Standard Energy

The standard energy change is the energy change when one mole of reactant is converted to one mole of product, it is the energy difference between reactants and products: ΔGo=Gproductso-Greactantso. The standard energy change defines the equilibrium position of a reaction through the following equation:

ΔGo=RTlnKEQ

  or  

KEQ=eΔGo/RT

For the simple reaction of A to B, the fraction of the system in state [A] is:

fA=[A][A]+[B]=11+KEQ

In a similar fashion, the fraction in state [B] is:

fB=[B][A]+[B]=KEQ1+KEQ

You can see from the above equations that if the energy of the products are equal to the reactants, then the equilibrium concentration of [B] will be equal to [A]. Mathematically, this can be shown as follows:

If the energy of [B] is equal to [A], then ΔGo = 0. Therefore

KEQ=1, and

fA=1/(1+1) = 0.5 and fB=1/(1+1) = 0.5

Question: What will happen to the relative concentrations of A and B if the energy of form A is lower than B, what will happen if the energy of form A is higher? Use the Learn-by-Doing tutorial below to find out.

Gibbs Free Energy

The formula for change in the Gibbs free energy in the reaction for the reaction direction [A] to [B] is:

ΔG=ΔG0+RTln[B][A]

Note that in this equation, the concentration of [A] and [B] are not necessarily at their equilibrium concentrations.

It can be shown that:

  • If ΔG less than 0 then the reaction is not at equilibrium and will proceed spontaneously in the forward direction, A to B.
  • If ΔG greater than 0 then the reaction is also not at equilibrium, but will proceed spontaneously in the reverse direction, B to A.
  • If ΔG=0 the reaction is at equilibrium and the concentrations of reactants and products will not change.

You can explore these relationships in the following tutorial.

In addition to predicting the direction of the reaction, the absolute value of ΔG, |ΔG|, is the amount of energy released when the concentrations of reactants and products change from their non-equilibrium values to their equilibrium values.

Question: What is the Gibbs free energy when the concentration of A is greater than its equilibrium value? In what direction will the reaction flow? From A to B or from B to A? Use the Learn-by-Doing tutorial below to find out.

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A reaction will be spontaneous in the forward direction if the Gibbs free energy for that reaction is less than zero. The same reaction, if run in the reverse direction, will have a positive Gibbs free energy and will therefore be non-spontaneous. If it is necessary to reverse the direction of this reaction to reverse the direction of the pathway, then the sign of the GIbbs free energy for the reverse reaction has to become negative. If you recall, the Gibbs free energy:

ΔG=ΔG0+RTln[B][A]

consists of two parts, the standard energy change, ΔGo, and a term that accounts for the non-equilibrium concentrations of [A] and [B]. Consequently, an unfavorable reaction with a positive Gibbs free energy can be made spontaneous by making the sum of these two terms negative by coupling the unfavorable reaction to a favorable, energy releasing one. The energy releasing reaction provides the necessary energy to change the sign of the Gibbs free energy from positive to negative.

There are two forms of coupling, both may be used to ensure a negative Gibbs free energy, direct and indirect coupling. Direct coupling reduces the standard free energy while indirect coupling reduces the second term of the equation. Both of these methods are described in more detail below.

  1. Direct coupling In this case a large negative ΔGo for the reaction is created by directly coupling the unfavorable reaction to a favorable one on the same enzyme. For example, the phosphorylation of glucose is very unfavorable if inorganic phosphate is used as the source of phosphate. The standard energy change of +14 kJ/mol makes the Gibbs energy positive. However, if the unfavorable reaction is coupled to the conversion of ATP to ADP within the active site of the enzyme hexose kinase the 30 kJ of energy released from ATP can be utilized to reduce the standard energy change such that the overall Gibbs free energy becomes negative. The overall reaction is the transfer of phosphate from ATP to glucose, with an overall standard energy change of approximately -15 kJ/mol. Direct coupling: Reaction 1 and 2 are hypothetical half-reactions that sum to give the complete reaction at the bottom of the image. In the actual reaction the enzyme glucose kinase transfers the phospate group directly from ATP to glucose; hydrolysis of the ATP does not occur. Note that the two half-reactions sum to give the complete reaction, both in terms of the compounds involved as well as the overall standard energy change.
  2. Indirect coupling. The Gibbs free energy can also become negative by either having a favorable reaction that preceeds the unfavorable one, or a favorable reaction that follows the unfavorable one. In the first case, the favorable preceeding reaction causes the concentration of the substrates for the following unfavorable reaction to be higher than equilibrium, making ΔG negative. For the case of a favorable following reaction, the concentration of the products of the unfavorable reaction are kept to a level that is smaller than the equilibrium concentration, again making ΔG negative. This type of coupling between reactions is referred to as indirect coupling because the coupling between favorable and unfavorable reactions occurs indirectly, via alteration of concentrations of reactants and products. Indirect coupling: In the absence of coupling (left) the conversion of [B] to [C] is uphill energetically, and therefore not favorable. If the preceeding step ([A] to [B]) is very favorable, the concentration of [B] increases to a level above its equilibrium level, which decreases the Gibbs free energy for step [B] to [C]. Alternatively, if the following step ([C] to [D]) is favorable, the concentration of [C] will be lower than its equilbrium level, which also decreases the Gibbs free energy for the step [B] to [C].
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There are thousands of different enzymes in any cell. Most enzymes bind a specific substrate, perform a simple chemical change on that substrate, and then release a product.

Each enzyme has a unique name. Usually, the name of an enzymes is systematic, but many exceptions exist. Almost all enzyme names have the suffix ase, indicating that they are an enzyme, for example kinase, lyase, phosphatase, etc. Usually, but unfortunately not always, the name of the enzyme is derived from the nature of the chemical change that it catalyzes. For example, an enzyme that oxidizes its substrate is referred to as a dehydrogenase because it removes hydrogens atoms during the oxidation process.

To further clarify the name of the enzyme, the name of the substrate or product is often included in the name. For example, the enzyme that oxidizes succinate is called succinate dehydrogenase. Keep in mind that most reactions in pathways are reversible, so the name may describe the reverse reaction. Lastly, in cases when enzymes bind more than one substrate, the name can also suggest the co-substrate. For examples, dehydrogenases will use NAD+ or FAD as co-substrates.

Important Classes of enzymes

Although there is a large number of enzyme catalyzed reactions in a cell, the following list describes most of the activities that are found in metabolic pathways.

Kinase. A kinase transfers a phosphate group from ATP to the substrate. Kinases are used when direct coupling is required to reduce the Gibbs free energy of the reaction.

Phosphorylation of glucose by glucose kinase. Note that the source of phosphate is ATP, not inorganic phosphate.

Alternatively, a kinase may be involved in regulation of enzymes by transferring a phosphate from ATP to a Serine, Threonine, or Tyrosine on the enzyme that is being regulated. The phosphorylated form of the enzyme may be active or inactive.

A protein kinase phosphorylates an enzyme, causing it to change its state from active to inactive or from inactive to active.

Phosphatase. A phosphatase removes a phosphate group by a hydrolysis reaction, producing inorganic phosphate. ADP or ATP are not involved in the reaction.

A phosphatase uses water to remove a phosphate group from its substrate. Phosphatases can act on small molecules, as shown above, or on phosphorylated proteins.

Dehydrogenase. As the name suggests, enzymes of this group transfer hydrogen atoms from the substrate to an electron acceptor, such as NAD+ or FAD . Therefore they are redox enzymes since removal of hydrogen atoms is the equivalent to removal of electrons. The name is applied to both oxidation and reduction reactions.

Succinate dehydrogenase, an example from the citric acid (TCA) cycle, is shown. FAD is the obligatory co-substrate, accepting electrons in this case. Note that this enzyme could also have been called fumarate dehydrogenase.

Isomerase. This class of enzymes rearrange functional groups on their substrates, releasing a product that has the same number of atoms as the substrate, but is an isomer of the original substrate. Unfortunately, the descriptive word "isomerase" is often omitted from their name.

Aconitase is an isomerase that functions in the citric acid cycle to convert citrate to isocitrate. Note that the composition of both substrate and product are the same, but the chemical structure is not.

Hydratase. This reaction type, which has a number of idiosyncratic names in different pathways, adds water to a double bond.

The addition of water to a double bond to produce an alcohol.

Synthetase. These enzyme are responsible for the synthesis of more complex molecules from simpler substrates. For example, ATP synthase generates ATP from ADP and inorganic phosphate, a process that is driven by a proton gradient across a membrane. Citrate synthase catalyzes the following reaction, which is the first in the citric acid (TCA) cycle.

Citrate synthase combines acetyl-CoA and oxaloacetate to form citrate. The source of energy in this case is the high energy thio-ester in acetyl-CoA.
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Glycolysis is the first pathway that occurs in the metabolism of carbohydrates. This pathway occurs in the cytosol of all cells, i.e. it is a highly conserved and ubiquitous pathway. Although many different disaccharides are broken down into monosaccharides and enter the glycolysis, we will focus on how glucose is metabolized in this section. The entry points of other sugars will be discussed in the module on integrated metabolism.

An overall summary of glycolysis is depicted below:

An overview of glycolysis. The intermediates in the pathway are shown near the top of the figure, along with a summary of energy consumption and production by the pathway. The molecular structure of these compounds can be seen in the learn-by-doing exercise below. ADP and NAD+ have been omitted in this diagram for clarity. The lower part of the figure shows the change in the carbon skeleton in the pathway. The step following F-1,6-P splits the 6 carbon fructose into two three carbon compounds, both of which proceed down the pathway to form pyruvate. The key regulatory step, the enzyme phosphofructose kinase, is indicated as PFK.

Key features of Glycolysis:

  1. Glucose, a six carbon hexose, is the input compound.
  2. Pyruvate, a three carbon keto-acid is the output. There is no loss of carbon in glycolysis, so two pyruvates are produced/glucose. Pyruvate is further oxidized in the TCA cycle.
  3. Two ATP molecules are produced/glucose. Note that the energy content of two ATP molecules is required to initiate the pathway. Four ATP (2/pyruvate) are produced later in the pathway. Consequently, the net yield is two ATP.
  4. Two NADH molecules are produced. A single oxidation step produces one molecule of NADH/pyruvate. The energy stored in NADH is extracted during electron transport.
  5. The key regulatory step is the addition of the second phosphate to fructose, by the enzyme phosphofructose kinase

Capture of Glucose by the Cell

Glucose from the outside of the cell is transported across the cell membrane by a multi-subunit protein called the glucose transporter. This enzyme catalyzes the diffusion of glucose across the membrane without the input of energy. Consequently, spontaneous flow of glucose into the cell can only occur if the concentration of glucose is lower in the cell than outside the cell. The Gibbs free energy for the transport of glucose is:

ΔG=RT ln[glucose]IN[glucose]OUT

Although this expression for the Gibbs free energy appears different than previous expressions, it is really the same equation. The "product" of the reaction is glucose inside the cell and the "reactant" is glucose outside the cell. The difference in standard free energy, ΔGo, is zero because the reactants and products are the same compound, differing only in their location. If the concentration of glucose outside the cell is higher than the concentration inside the cell, then the Gibbs free energy is negative (the ln of a number less than one is negative) and glucose will spontaneously enter the cell. If the glucose concentration in both compartments are the same, then Δ=0 and there is no net movement of glucose across the membrane. If the concentration of glucose inside the cell exceeds the concentration outside then Δ>0 and the reverse reaction, the net movement of glucose out of the cell, will be spontaneous.

The levels of glucose in the blood vary considerably at times. We will discuss how glucose levels are regulated in the section on integrated metabolism. The focus here is to understand how the entry of glucose into the cell is spontaneous, even when the levels of glucose in the blood can drop to low values. How is it possible to maintain a negative Gibbs free energy for glucose transport into the cell, even if there are low levels of glucose outside the cell?

Question: Use the tutorial below to discover how glucose spontaneously enters the cell, even if the intracelluar concentration exceeds the concentration outside the cell.

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Glycolysis

The following learn-by-doing activity allows you to investigate the chemical and energetic changes that occur in glycolysis. Once you open up the page, explore it using the embedded questions to prepare you for the self-assessment at the end of this module.

Question: As you move through the glycolysis pathway, determine which steps become spontaneous by direct coupling, which by indirect coupling, and how energy is captured from steps that release large amounts of energy.
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Anaerobic Glycolysis

Under anaerobic (oxygen limited) conditions, which occurs in the muscle tissue during vigorous activity, the NADH produced in glycolysis cannot be reoxidized to NAD+ by electron transport because there is insufficient oxygen to accept electrons. Under these conditions, the cell runs out of NAD+ and glycolysis will halt and the cell can no longer produce ATP.

The levels of NAD+ can be restored by using pyruvate as the electron acceptor. In mammals, lactate is the product of this reaction. In yeast, it is alcohol and the process of anaerobic glycolysis is referred to as fermentation. The reduction of pyruvate will oxidize NADH to NAD+, allowing glycolysis to resume.

Anaerobic metabolism. Pyruvate can serve as the electron acceptor for NADH. The reduction of pyruvate to lactate in mammals, or ethanol in yeast, regenerates the NAD+ required for glycolysis to operate.

The lactate that is produced by active muscle tissue is transported to the liver. When oxygen becomes available, the lactate is reoxidized to pyruvate and it can then be used for the synthesis of glucose.

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Catabolic Process of the TCA Cycle

The tricarboxylic acid (TCA) cycle, also known as the Krebs cycle or the citric acid cycle, is a central pathway in the metabolism of all organisms. Not only is this pathway the next step for the oxidation of glucose and fatty acids, it also plays a key role in amino acid metabolism. All carbons that enter the TCA cycle, regardless of their source, are fully oxidized to carbon dioxide. The electrons that are released from these oxidations are stored on NADH and FADH2 for subsequent processing by the electron transport chain.

Entry of carbohydrates into the TCA cycle occurs by the transport of pyruvate from the cytoplasm into the mitochondrial matrix. There, the pyruvate undergoes a process called oxidative decarboxylation to produce a key intermediate in metabolism, acetyl-CoA, which enters the TCA cycle.

Entry of fatty acids into the TCA occurs directly into acetyl-CoA. During the process of fatty acid oxidation (beta-oxidation) which also occurs in the mitochondrial matrix, two-carbon fragments are progressively released from fatty acids and incorporated into acetyl-CoA.

Entry of carbohydrates and fats into the TCA cycle. Oxidative decarboxylation of pyruvate produces acetyl-CoA, releasing one CO2 and NADH. Fatty acid oxidation yields acetyl-CoA directly. Acetyl-CoA is a high-energy thioester that enters the citric acid cycle.

The first step of the TCA cycle utilizes the high-energy thioester in acetyl-CoA to drive the addition of an acetate group to oxaloacetic acid to produce citrate.

In the first step of the TCA cycle acetyl-CoA donates an acetate group to oxaloacetate, forming citrate. Note that the ketone group on oxalacetate has become an alcohol in citrate.

The remaining steps in the TCA cycle convert citrate back to oxaloacetate. This process produces:

  • 2 CO2, from oxidative decarboxylations.
  • 3 NADH, all from oxidation of alcohols to ketones.
  • 1 FADH2, from the oxidation of an alkane to an alkene
  • 1 GTP, from the hydrolysis of a thio-ester.

Given the above information, your task is to deduce the series of reactions that convert citrate back to oxaloacetate using the following learn-by-doing activity.

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Anabolic Role of the TCA Cycle.

Intermediates of the TCA cycle are utilized in the following synthetic pathways:

  • Fatty acids and steroids from acetyl-CoA.
  • Amino acids aspartate, asparagine, lysine, isoleucine, methionine from oxaloacetate.
  • Amino acids glutamate, glutamine, proline, arginine from alpha-ketoglutaric acid
  • Porphyrin, a precursor to heme, from Succinyl-CoA

What major metabolite is missing from this list? Carbohydrates! It is not possible for mammals to use carbon atoms derived from the TCA cycle to synthesize glucose. How glucose is synthesized from pyruvate will be discussed in the module on integrated metabolism.

If compounds in the TCA cycle are used to synthesize other compounds, such as amino acids, the TCA cycle will eventually halt due to depletion of oxaloacetate. Consequently it is necessary to "fill up" the TCA cycle with an "anaplerotic" reaction. In this case, pyruvate is converted to oxaloacetate by the enzyme pyruvate carboxylase:

Anaplerotic reaction that generates oxaloacetate from pyruvate. This reaction replenishes the carbons in the TCA cycle, allowing it to continue to operate under anabolic conditions.
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The production of ATP from "high-energy" electrons on NADH and FADH2 occurs in two steps, electron transport followed by ATP synthesis. The energy obtained from electron transport is stored as a pH gradient across the inner mitochondrial membrane. The combination of a concentration difference of hydrogen ions and a voltage difference accross this membrane is often referred to as an electrochemical gradient. The electrochemical gradient is a form of potential energy that is utilized by the enzyme ATP synthase to convert ADP to ATP. The coupling between the proton gradient and the chemical synthesis of ATP, was originally proposed by Dr. Peter Michell in 1961 as the chemiosmotic hypothesis. His theory proved to be true, leading to a Nobel prize for his work in 1978.

Electron Transport

The electron transport chain consists of four multi-protein complexes that are contained within the inner mitochondrial membrane. These complexes remove electrons from NADH or FADH2 molecules that were generated by oxidative processes in glycolysis and the TCA cycle. These high energy compounds ultimately deposit their electrons on oxygen, forming water. The energy that is released by electron transport is stored as a proton gradient across the inner mitochondrial membrane. This proton gradient is used to drive the synthesis of ATP.

The hilighted region focusing in on the Electron Transport Chain, part of the complex process of metabolism.

Various compounds are used by the electron transport chain to carry electrons. These components include:

  • FAD/FADH2, is a cofactor that is tightly bound to the enzymes in the electron transport complexes.
  • Iron-sulfur centers in the electron transport complexes shuttle electrons by alternating between Fe+2 and Fe+3.
  • Coenzyme Q is an organic non-polar electron carrier that is dissolved within the membrane lipids of the inner mitochondrial membrane. It carries electrons between complex I and III and complex II and III.
  • Cytochrome C is a heme containing protein that is very similar in structure to myoglobin. It is a water soluble protein found in the inter-membrane space. Cytochrome C carries one electron at a time from complex III to complex IV.

The organization of the four complexes and electron carriers in the electron transport chain are illustrated in the following diagram:

The electron transport chain is contained within the inner membrane of the mitochondria. In addition to the four protein complexes, the location of electron carriers coenzymeQ and cytochrome C are also indicated. Complex I accepts electrons from NADH and complex II accepts electrons from succinate. Complex IV is responsible for transferring the electrons to the final electron acceptor, oxygen. Selecting the button labeled NADH will show the flow of electrons from NADH to complex IV. The button labeled FADH2 will show the path of electrons from succinate to FADH2 in complex II (succinate dehydrogenase), followed by movement of these electrons to complex IV.

NADH is oxidized by the electron transport chain by the transfer of the two electrons from NADH to complex I. These electrons are then transferred to coenzyme Q, followed by transfer to complex III. The electrons are then carried by cytochrome C to complex IV, where the electrons are used to reduce oxygen to water. The energy that is released by the transfer of electrons from NADH to water is used to transport a total of 10 protons across the inner membrane. For every pair of electrons, four protons are transported by complex I, four by complex II and two by complex IV.

The electrons from FADH2 are first processed by complex II. Complex II is actually succinate dehydrogenase from the TCA cycle. The electron acceptor for succinate, FAD, is tightly bound to the enzyme. Consequently, it is more correct to consider that complex II processes the electrons from succinate, first passing them to FAD and then via iron-sulfur centers to coenzyme Q. The two electrons then follow the same path as those from NADH2, from coenzyme Q to complex III, and then to complex IV via cytochrome C, finally to oxygen to produce water. Since electron transport through complex II does not result in the transport of protons only a total of 6 protons/2 electrons are transported across the inner membrane when succinate is the initial electron donor.

In summary:

  • Electrons from NADH and FADH2 are transferred to oxygen, generating water.
  • Electron transport is spontaneous and releases energy. This energy is stored by transporting protons across the inner membrane.
  • Oxidation of NADH results in 10 protons transported across the inner membrane.
  • Oxidation of FADH2 results in 6 protons transported across the inner membrane.
This link will take you to a movie providing an alternate representation of the electron transport process.

ATP Synthesis

The energy that has been stored in the proton gradient across the inner mitochondrial membrane cannot be easily utilized by other processes in the cell. It must be converted to a more usable source, such as ATP. The enzyme ATP synthase is responsible for converting the energy stored in the proton gradient to ATP. This enzyme is found in the inner mitochondrial membrane and it projects into the matrix.

The mechanism of ATP synthase is such that the enzyme generates one ATP molecule every time 3 protons are transferred back to the matrix from the inter-membrane space. Since the standard energy for the formation of ATP from ADP and Pi is +30 kJ/mol the transport of three protons must release at least 30kJ/mol in order to provide enough energy to synthesize ATP. The amount of energy that is stored in the protein gradient can be obtained by calculating the Gibbs free energy. If the products are defined as the protons that have been transported to the matrix, the formula is:

ΔG=RT ln[H+]IN[H+]OUT+ZFΔΨ

where Z is the change on the transported particle, F is Faraday's constant (96,494 Coulomb/mol) and ΔΨ is the voltage difference across the membrane:

ΔΨ=VINVOUT

The Gibbs free energy consists of two terms. The first indicates the energy change due to the difference of proton concentration across the membrane. Its form is analogous to the formula give for the transport of glucose across the membrane in glycolysis. The second term accounts for the fact that the energy of a charged particle depends on the voltage. If there is a voltage difference across the membrane then the energy of the proton will depend on its location.

Typical proton concentration differences across the inner membrane are approximately 10 fold, with the outside being more acidic. In addition to the concentration difference, there is also an approximately 100 mV voltage difference across the membrane, with the inside being more negative. Therefore, the Gibbs free energy change under these conditions at 300 K is:

ΔG=R(300)ln110+1×96494×(0.1)=5.79.6 kJ/mol=15.3 kJ/mol

Therefore the transport of 3 protons will release 45.9 kJ/mol, which is more than sufficient to generate a single ATP.

Mechanism of ATP Synthase

The synthesis of ATP utilizing the proton gradient as a source of energy is an example of direct coupling. The energy released as the protons flow through the enzyme cause a conformational change in the protein that causes the formation of ATP from bound ADP and inorganic phosphate.

The enzyme consists of two separable subunits. The Fo subunit is within the membrane and is responsible for the transfer of protons through the membrane. The F1 subunit extends into the mitochondrial matrix and is responsible for the synthesis of ATP. The F1 subunit is composed of three α subunits and three β subunits. These six subunits from a spherical structure where the α and β subunits alternate. The γ subunit extends from the Fo domain through the center of the α-β sphere. The γ subunit rotates 120 degrees every time three protons flows through the Fo domain. The α-β sphere is prevented from rotating along with the γ subunit by the b-subunit, which anchors the α-β sphere to the membrane.

The conformation of the α-β subunits is affected by the relative position of the γ subunit. Since the gamma subunit rotates 120o, there are three possible conformations of the α-β subunits:

  • A conformation that has low affinity of nucleotides, i.e. neither ATP or ADP bind.
  • A conformation that has high affinity for ADP plus inorganic phosphate.
  • A conformation that has high affinity for ATP, i.e. ATP is more stable in this conformation than ADP.

The cycle of ATP synthesis is as follows:

  1. ADP and inorganic phosphate bind to the subunits that have high affinity for these compounds.
  2. A three protons flow through the Fo domain, causing a 120o rotation of the γ-subunit.
  3. The rotation of the γ-subunit causes a conformational change in the α-β subunits.
  4. ATP is more stable in the new conformation of the α-β subunits, consequently the bound ADP and inorganic phosphate is spontaneously converted to ATP.
  5. Another three protons flow through the Fo domain, causing another 120o rotation of the γ subunit.
  6. This rotation causes an additional conformational change of the α-β subunits, generating the conformation that has low affinity for ATP and ADP, thus the newly synthesized ATP is released.
  7. A three additional protons flows through the Fo domain, causing another rotation of γ subunits. This restores the system to the starting conditions.

Complete rotation of the γ subunit requires the transfer of 9 protons across the membrane. This generates a total of 3 ATP molecules since there are three ADP/ATP binding sites on the F1 domain, one associated with each α-β subunit. Consequently, only three protons are required to synthesize one ATP.

Click the green arrow or PLAY button to play the animation.
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The storage and metabolism of glucose is controlled at the organ level by hormones. The hormones glucagon and insulin are secreted by the pancreas during periods of low or high blood sugar, respectively. Glucagon causes the liver to produce glucose from the storage polysaccharide glycogen or to synthesize glucose from pyruvate using the pathway gluconeogenesis. The released glucose enters into the blood and travels to muscle for oxidation by glycolysis leading to energy production. In contrast, insulin instructs the liver cell to store the excess glucose in the blood.

The hormone epinephrine,which is produced by the central nervous system in response to dangerous situations, evokes the same response as glucagon, the release of glucose from the liver.

Hormonal control of glucose metabolism. Glucagon and insulin are pancreatic hormones that regulate blood sugar levels. Epinephrine (adrenaline) is produced by the adrenal gland in the central nervous system in response to dangerous situations. Glucagon and epinephrine instruct the liver cell to produce glucose by release from glycogen or by synthesis from pyruvate via the synthetic pathway gluconeogenesis. The glucose enters the blood and is oxidized in muscle cells to produce energy. Insulin is secreted when the blood glucose level is high, instructing the liver to store glucose in glycogen or, if necessary, to oxidize it to produce energy.

These hormones do not directly affect the enzymes involved in glucose metabolism and storage. Rather they bind to membrane receptors on the surface of the cell and evoke a conformational, or allosteric, change in the receptor, transmitting the signal to the inside of the cell.

Transmitting the Signal to the Inside of the Cell
The hormones glucagon, epinephrine, and insulin all bind reversible to receptors on the cell surface. The binding of glucagon and epinephrine ultimately lead to protein phosphorylation by the activation of protein kinases while the binding of insulin ultimately leads to activation of protein phosphatases which remove the phosphate groups from enzymes.
Click the green arrow to play the animation.

The phosphorylation of enzymes during periods of glucose demand or the dephosphorylation of enzymes when there is surplus glucose results in the correct biological response of the liver cell. Phosphorylation results in the activation of enzymes that cause the release of glucose from glycogen and also results in the synthesis of new glucose by the anabolic pathway gluconeogenesis. Dephosphorylation of enzymes causes the activation of enzymes involved in the storage of glucose in glycogen and can also result in the activation of glucose oxidation for ATP production.

Since the protein kinases and phosphatases that are involved in the activation of enzymes are enzymes themselves, amplification of the initial hormone-receptor signal occurs. This is especially true in the case of the hormones glucagon and epinephrine where additional amplification occurs due to the production of a large number of cAMP molecules per signalling event.

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Glycogen is a polysaccharide that is used to store glucose in both liver and muscle cells. In plants, amylopectin serves the same role. Glycogen consists of glucose residues linked together in a linear chain with α-(1-4) linkages. Multiple chains are joined together by the formation of α-(1-6) linkages, giving rise to a highly branched polymer. The principle reason for the storage of glucose as glycogen is to reduce the osmotic effects that would otherwise occur from the high concentration of glucose in cells.

Glycogen. The overall structure of a small glycogen molecule is shown on the left. An enlarged portion of the molecule is shown on the right.

When needed, glucose is released from glycogen by a reaction catalyzed by the enzyme glycogen phosphorylase. This enzyme breaks an α-(1-4) linkage at the end of glycogen polymer by the addition of an inorganic phosphate. The product of this reaction, glucose-1-P, can enter glycolysis after conversion to glucose-6-P. Note that this entry point for glucose skips the first step in glycolysis, avoiding the need to activate glucose using ATP.

Glycogen degradation occurs by the cleavage of an α-(1-4) linkage with inorganic phosphate, catalyzed by the enzyme glycogen phosphorylase. This is reaction occurs spontaneously, no external energy is required.

For storage, glucose is added to the free 4-hydroxyl groups of the glycogen molecule, elongating the linear chains. Since the release of glucose by phosphorolysis is spontaneous, the direct addition of glucose to glycogen must be energetically unfavorable. Consequently, the synthetic reaction requires coupling to an energy releasing reaction to become spontaneous. The addition of glucose to glycogen proceeds in two steps. First, glucose-1-phosphate reacts with UTP to produces a high energy intermediate, UDP-glucose, which in turn is used by the enzyme glycogen synthase to add glucose to glycogen.

Glycogen synthesis. The incorporation of glucose into glycogen requires two steps. In the first (I) activation of glucose occurs using the energy from UTP to form UDP-glucose. Remarkably, the formation of UDP-glucose occurs with a free energy change of nearly zero. The overall Gibbs free energy of the reaction becomes negative due to the energy released when pyrophosphate is hydrolyzed to inorganic phosphate. In step II, the high energy compound UDP-glucose is used to spontaneously add glucose to glycogen. The net energy cost of adding a glucose to glycogen is two ATPs, one to form glucose-1-phosphate and one to form UTP from UDP. Since glucose is released from glycogen in a phosphorylated state, glucose-1-phosphate, the net cost to store glucose in glycogen is one ATP/glucose.

Regulation of Glycogen Synthesis and Degradation: Glycogen synthesis and degradation is controlled by the phosophorylation state of the enzymes. The phosphorylation levels of proteins within the cell are directly controlled by response to hormones, as discussed above.

Glycogen regulation by phosphorylation of enzymes. Glycogen phosphorylase is active when phosphorylated. In contrast, the phosphorylation of glycogen synthase inactivates the enzyme. Dephosphorylation leads to a reversal in the regulation; inactivation of glycogen phosphorylase and activation of glycogen synthase.
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Glucose Synthesis - Gluconeogenesis

During glycolysis glucose is oxidized to pyruvate. The reverse reaction, the synthesis of glucose from pyruvate occurs in the gluconeogenesis pathway. The major source of pyruvate in the liver is lactate that was produced by active muscle during anaerobic metabolism.

Since the process of converting glucose to pyruvate via glycolysis is energy releasing, the reverse reaction must require energy. Most of the steps in the gluconeogenesis pathway have a Gibbs free energy of nearly zero, and are easily reversed by slight changes in the concentrations of products and reactions. However, there are three steps in glycolysis that occur with such a large decrease in energy that they cannot become spontaneous in the reverse direction. These steps are:

  1. The formation of glucose-6-P from glucose, coupled to ATP hydrolysis.
  2. The formation of fructose-1-6-P from fructose-6-P, coupled to ATP hydrolysis.
  3. The formation of pyruvate from phosphoenolpyruvate (PEP), releasing sufficient energy to produce ATP.

Since the first two reactions use ATP to drive an otherwise unfavorable reaction, the phosphorylation of sugars, the simpler reverse reaction, hydrolysis, will be favorable and spontaneous. Consequently, in gluconeogenesis these two steps are catalyzed by phosphatases instead of kinases. The third reaction in glycolysis, phosphoenol pyruvate (PEP) to pyruvate, has such a large decrease in energy that it cannot be easily reversed and must be accomplished by a different method.

In gluconeogenesis the conversion of pyruvate to PEP in gluconeogenesis occurs by in two steps:

  1. The enzyme pyruvate carboxylase catalyzes: pyruvate + HCO3- + ATP → oxaloacetate + ADP + Pi
  2. The enzyme PEP carboxykinase catalyzes: oxaloacetate + GTP → phosphoenolpyruvate + GDP + CO2.

Therefore a total of two "high energy" phosphate bonds (ATP/GTP → ADP/GDP +Pi) are required to reverse this step.

The complete pathways for glucolysis and gluconeogenesis are shown below:

Glycolysis and Gluconeogenesis. The left part of the figure shows glycolysis, the right side shows gluconeogenesis. Enzymatic steps that are accomplished by different mechanisms (and enzymes) in each pathway are highlighted in yellow. The hydrolysis of phosphate from fructose 1,6-P and glucose-6-P is spontaneous, consequently the kinases used in glycolysis are replaced by phosphatases in gluconeogenesis. The conversion of pyruvate to PEP follows a completely different route in gluconeogenesis than in glycolysis. The enzymes whose names are colored red, phosphofructose kinase (PFK) and fructose 1,6 bisphosphatase, catalyze important regulated steps in each pathway.
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Glycolysis and gluconeogenesis are regulated at two levels. First, at the cellular level, the regulatory enzymes are sensitive to the energy levels of the cell. Second, at the organ level, the enzymes are regulated by hormones. However, unlike glycogen metabolism, this regulation is not via protein phosphorylation but by changes in levels of the allosteric regulatory compound fructose 2,6 bisphosphate (F26P). The levels of F26P are indirectly regulated by hormones by virtue of the fact that the activity of the enzymes that make and degrade F26P are a affected by protein phosphorylation.

Regulation by Energy Sensing. Cells have a relatively constant amount of adenosine nucleotides, i.e. the sum of the concentrations of AMP, ADP, and ATP are relatively constant. When the cell is consuming energy, ATP is being hydrolyzed to ADP or to AMP. Consequently, high levels of AMP and ADP indicate that the cell needs to activate glycolysis to restore the levels of ATP. If ATP levels are high, then glycolysis should be turned off. The excess ATP can be used to synthesize glucose and other molecules if needed.

Cellular energy levels are high when ATP or NADH levels are high. Cellular activities convert ATP to ADP and AMP. ATP levels can be replenished by oxidation of NADH by electron transport and ATP synthesis. When both NADH and ATP levels are low, the cell has low energy reserves.

The enzymes phosphofructose kinase and fructose-1,6-bisphosphatase are under allosteric control by adenosine nucleotides:

High ATP

HIgh AMP (low ATP)

Note that these two enzymes are regulated in a coordinated manner such that only one pathway, glycolysis or gluconeogenesis, is turned on at one time.

Regulation by Hormonal Control. The compound fructose -2,6 bisphosphate (F26P) is an allosteric activator of PFK. F26P also inhibits fructose bisphosphatase, the key regulatory enzyme in gluconeogenesis. The levels of F26P are controlled by hormones via protein phosphorylation levels. When protein phosphorylation levels are high, F26P is degraded, when phosphorylation levels are low, F26P is synthesized.

Regulation of fructose-2,6-bisphosphate (F26P) levels by hormones. Glucagon and epinephrine cause phosphorylation of enzymes. The enzyme that degrades (F26P) is activated when phosphorylated, consequently F26P levels drop when either of these hormones are present. The reverse occurs when insulin is present, the kinase becomes activated and F26P levels rise. Note that F26P is generated from fructose-6-phosphate (F6P), which is also an intermediate in glycolysis. Since the enzyme that synthesizes F26P has the same function as PFK in glycolysis, adding a phosphate to fructose-6-P, it is called PFK-2.

The regulation of glycolysis and gluconeogenesis by energy sensing and indirect hormonal control is summarize below.

Regulation of PFK in glycolysis and fructose 1,6 phosphatase in gluconeogenesis by energy sensing and fructose-2,6-bisphosphate levels. Note that when one pathway is activated, the other is inhibited.

The coordinated regulation of glycolysis and gluconeogenesis dovetails perfectly with the direct hormonal control of glycogen metabolism to insure the liver cell responds appropriately to hormonal signals, i.e. the production and release of glucose when glucagon or epinephrine are present, or the storage and, if required, the oxidation of glucose when insulin is present.

The left panel shows the steps that occur when there is a demand for glucose from the liver, as signaled by the hormones glucagon or epinephrine. The end result is release of glucose from glycogen and the activation of gluconeogenesis if there are sufficient ATP reserves. The right panel shows the steps that occur when the liver (and muscle) are to store excess glucose, as signaled by the hormone insulin. The end result is the synthesis of glycogen and, if the cell requires energy (ATP), the activation of glycolysis.
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Obesity and the Metabolism of Fats, Amino acids, and Complex Sugars

Fats are stored in cells as triglycerides, the condensation product of three fatty acids with the three carbon polyalcohol glycerol. Hormonal stimulation by glucagon or epinephrine activates lipases which release the fatty acids from the glycerol. The fatty acids are activated for oxidation in the cytosol by the addition of an coenzyme A group to the carboxylic acid, forming acyl-CoA. After activation, the activated fatty acid is transported to the mitochondria where it is subject to a pathway called beta-oxidation. This process releases acetyl-CoA molecules from the fatty acid, generating NADH and FADH2. The acetyl-CoA enters the TCA cycle for further oxidation.

Fatty acids are stored in the cytosol as triglycerides. Fatty acids are released from triglycerides by the action of lipases. To begin the oxidation process, the fatty acid is activated by converting the carboxylic acid to thioester to coenzymeA, generating acyl-CoA. The acyl-CoA is transported into the mitochondrial matrix where oxidation occurs. Fatty acids are stored in the cytosol as triglycerides. Fatty acids are released from triglycerides by the action of lipases. To begin the oxidation process, the fatty acid is activated by converting the carboxylic acid to thioester to coenzymeA, generating acyl-CoA. The acyl-CoA is transported into the mitochondrial matrix where oxidation occurs.

Fatty acids can also be synthesized from acetyl-CoA by an energy requiring process. As with glycolysis/gluconeogenesis many of the same enzymes are utilized in both pathways. However, highly exergonic reactions in the degradative pathway are accomplished by a different mechanism in the synthetic pathway.

Complex carbohydrates are converted to monosaccharides and enter glycolysis at a number of different points. Most monosaccharides enter glycolysis above the step catalyzed by phosophofructose kinase (PFK). Consequently, the degradation of practically all sugars is controlled by this key regulatory enzyme.

Metabolism of complex carbohydrates. Glycogen releases glucose as glucose-1-P which can be readily converted to glucose-6-P and enter glycolysis. Sucrose, or table sugar, is broken down into glucose and fructose. The fructose is phosphorylated and enters glycolysis above the PFK step. Lactose, the major sugar in milk, provides one glucose that enters glycolysis directly and one galactose. The chiral center at the fourth carbon of galactose is then inverted, creating glucose.

Although the metabolism of most amino acids is a complicated multi-step process, a number of amino acids enter the main oxidative pathways by a single transaminase reaction. In the transaminase reaction a ketone group is replaced by an amino group. For example, the amino acid alanine is converted to pyruvate by the following transaminase reaction:

Alanine can be reversibly converted to pyruvate by transamination.

Why do Humans Get Fat?

A key feature of human metabolism is the fact that the conversion of pyruvate to acetyl-CoA is irreversible. Consequently, once ingested carbon from any source, fats, protein, or carbohydrates, is converted to acetyl-CoA it has only two fates:

  • Entry into the TCA cycle for oxidation and the production of energy.
  • Storage as triglycerides, in specialized adipose tissue.

The TCA cycle is regulated by energy sensing. When the levels of the high energy compounds ATP or NADH become elevated the TCA cycle halts - there is no need for the cell to produce energy. Under these conditions, any excess acetyl-CoA is stored as fat, regardless of the source of the acetyl-CoA. The ingestion of excess amounts of proteins or carbohydrates result in the generation of fat.

Fats are a less desirable form of energy storage since the production of energy from fats is slower than the production from glucose. In addition, a number of tissues in the body, in particular the brain, use glucose as their principal energy source. Consequently, the enzyme that converts pyruvate to acetyl-CoA is tightly regulated.

Pyruvate dehydrogenase is inhibited by:

  • Product inhibition by NADH and acetyl-CoA.
  • Phosphorylation of the enzyme causes inactivation.

Consequently, pyruvate will not be converted to acetyl-CoA if there is excess NADH or acetyl-CoA. Furthermore, the hormones glucagon and epinephrine, which evoke protein phosphorlation, prevent the loss of pyruvate to acetyl-CoA such that pyruvate can be used for the synthesis of glucose.

Fats are synthesized from excess amino acids and sugars.

Although pyruvate dehydrogenase is well regulated, it is clear by observation of the human population that the flow of carbon from pyruvate to acetyl-CoA to fatty acids still occurs.

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This page is dedicated to instructions about the Activities.

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Many pages of this course contain formulas which require that your browser supports displaying MathML. You should run "Test and Configure My System" from your My Courses page to make sure your browser supports the tools the course uses.

Types of Activities

This course has many interactive activities that you are encouraged to complete. You can repeat many of the activities as necessary. General instructions for the use of the tools will always be available in the Glossary Unit: Instructions for Activities.

  • "Learn By Doing" and "Explore further" These links take you to exercises you can repeat multiple times. They will usually be interactive and help you to understand the concept being discussed. These activities are "low stakes." You will not be graded on these activities.
  • "Did I Get This" links take you to self-assessment questions. These exercises are graded for your information only; the grade is not recorded in the grade book. Many of the questions will contain hints and feedback to help you guide your learning and review of the material. You may take these quizzes as many time as you like.
  • "Many Students Wonder" are activities that provide additional detail or material. These materials supplement the ideas being presented with articles showcasing current research, items in the news, or other interesting tidbits.
  • "Quiz" This is a graded assessment. The grade will be recorded in the gradebook, and you can take these quizzes only one time. You should take these quizzes only after you have mastered the other activities such as the "Learn By Doing" and "Did I Get This." These assessments are not available in the Open and Free version of the course.
  • "My Response" This activity serves two functions. The My Response buttons in the content follow questions posed in the text. You should treat these questions as practice for the exam. Your first response will be saved for you to view in the future. The responses are not graded but are visible to the instructors and may be used in classroom dicussion. The second use of the "My Response" button is at the end of a lesson just before a graded exam. This activity is you chance to send questions to the instructors. The questions can be for clarification of the material or for something you find confusing. The questions can also be for further information. The questions you pose are visible to the instructors may be used for in class discussion.

Flash Interaction

Any Flash movie can be zoomed.

  • Mac users: "Control" click on the Flash movie and select the "Zoom In" option in the menu.
  • PC users: Right-click on the Flash movie for the menu of options.

Jmol 3-D Molecule Viewer

Jmol is an open-source Java viewer for chemical structures in 3D. Jmol returns a 3D representation of a molecule that may be manipulated in a number of ways.

  • Mac users: Click and drag to rotate the molecule. Option drag to zoom in or out. "Control" click on the Jmol for the menu of options.
  • PC users: Click and drag to rotate the molecule. Alt drag to zoom in or out. Right-click on the Jmol for the menu of options.

FlashMol 3-D Molecule Viewer

FlashMol allows you to see chemical structures in 3-D and to rotate the structure in all three dimensions.

Click an hold an area of the 3-D molecule and drag the mouse cursor in the direction you want the molecule to move. Compare the 3-D molecule to the text representation.

In the Functional Groups Activity you can find many examples of 2-D and 3-D representations.

The following is the list of terms defined in this course.

Aldose
(Definition)
A category of simple carbohydrates where the number 1 carbon (the top carbon) contains the carbonyl that is flanked by a hydrogen and a carbon thus making this an aldehyde. Refer to page.
Allosteric Binding
(Definition)
Allosteric binding causes conformational changes in an enzyme that can either inhibit or activate the enzyme. Refer to page.
Catalyst
(Definition)
A catalyst is a participant in a chemical reaction that speeds up the reaction but is not consumed itself. Enzymes are biological catalysts that mediate the conversion of substrate to product, by lowering the activation energy of the reaction. Enzymes re not consumed or modified during the process. Enzymes are generally made of proteins but nucleic acid enzymes, ribozymes, have also been discovered. Refer to page.
Chirality
(Definition)
When identical groups attached to a carbon are arranged in multiple ways such that two of the resulting structures are non-superimposable, they are mirror images of each other. Refer to page.
Covalent bond
(Definition)
Covalent bonds represent the sharing of the electrons (negatively charged subatomic particles between atoms.) The number of covalent bonds that can form is dictated by the number of unpaired electrons in the outer valence shell of the atom. Refer to page.
De novo synthesis
(Definition)
De novo is new synthesis from simple molecules such as deoxynucleotides in this case without a starting place - the primer. Refer to page.
Electronegativity
(Definition)
The tendency of an atom to attract electrons to itself. Electronegativity increases as one moves from left to right, across the periodic chart. Because the electronegative atoms have the potential to attract electrons, i.e., the electrons spend more time on the electronegative atom, a molecule containing an electronegative atom will have partial negative charge associated with that atom (as indicated by δ-). The the bonding partner in the covalent bond becomes partially positively charged (as indicated by δ+ ). Refer to page.
Eukaryotes
(Definition)
An organism with complex cells with distinctive traits such as a nucleaus, membrane-bound organelles, a cytoskeleton, and the presence of introns in genes. Refer to page.
Furanose
(Definition)
Cyclic sugars that contain a five membered ring are called furanoses. Refer to page.
Genome
(Definition)
the complete DNA sequence of one set of chromosomes from an organism. Refer to page.
Hydrogen Bond
(Definition)
The attraction of an electronegative atom for a hydrogen that is covalently bonded to another electronegative atom. This involves the attraction of a hydrogen with a partial positive charge to an atom with a partial negative charge. However, only hydrogens covalently bonded to an electronegative atom can participate in hydrogen bonding. Refer to page.
Hydrophilic
(Definition)
We need a good definition and image here Because polar molecules are generally water soluble, they are referred to as being hydrophilic, or water-loving. Refer to page.
Hydrophobic
(Definition)
Hydrophobic, or water-fearing, molecules do not interact with water and are characterized by a complete lack of electronegative atoms. In aqueous solutions the hydrophobic molecules are driven together to the exclusion of water. Refer to page.
Ionic Bond
(Definition)
An ion is an atom or molecule which has lost or gained one or more electrons, making it positively or negatively charged.We need a complete definition Ionic bonds form between oppositely charged atoms. No electron sharing or transfer occurs. The atoms are attracted to each other due to their opposite charges. Refer to page.

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Ketose
(Definition)
A carbohydrate with a carbonyl at the number 2 carbon (the center carbon) that is flanked by carbons on both sides, thus making this carbonyl a ketone, is a ketose. This is a polar, hydrophilic, water-soluble molecule. Refer to page.
Polymer
(Definition)
These are large molecules consisting of repeating structural units, or monomers, connected by covalent chemical bonds. Refer to page.
Prokaryotes
(Definition)
A unicellular organizm lacking certain complex cell features, such as a membrane-bound nucleus and gene introns. Refer to page.

Biologists have different ways of representing chemical structures. Each type conveys different information.

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Use this as a reference for the important functional groups and representative molecules for each group. You can select individual groups or select by property. Inside each functional group there are multiple examples of the group in actual molecules. The molecules are represented as text and in 3-D.

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The following is the list of tutorial animations explaining various complex biological processes.

Biological Membranes Biological membranes are dynamic structures composed of a diverse set of phospholipid molecules and proteins. This tutorial explores some of the properties of the membranes. Refer to page.
Phase Transition Phospholipids bilayers undergo a cooperative phase transition or melting that is similar to protein denaturation. The high degree of cooperativity is due to extensive interactions between the non-polar acyl chains in the center of the bilayer. The overall structure of the lipid bilayer is not changed by this transition. However, the disorder of the non-polar hydrocarbon chains increases dramatically after melting. Refer to page.
Glucose Transport The tutorial shows how the glucose transporter molecule is structured with a spiral channel that allows the glucose molecules to passively navigate the channel and move through the membrane. Refer to page.
Signal Transduction This animation illustrates how binding of a ligand to its receptor ultimately leads to the production of high levels of cAMP, an important second messenger in the signal transduction pathway. Refer to transport page. [ Refer to signaling in metabolism page ]
Lactose Permease Transporter The Escherichia coli lactose permease is an example of secondary active transport (Campbell, p. 210). This enzyme is similar in structure to others in the major facilitator superfamily (MFS) of transporters. More than 1000 examples of MFS transporters have been identified in the genomes of bacteria, plants, and animals. Refer to page.
Receptor Mediated Endocytosis Many macromolecules are taken into the cell by a process described as endocytosis. The macromolecules do not pass through the membrane directly to the cytoplasm but instead are taken up by the cell, processed by cell and then delivered to the cytoplasm. For many of the endocytic processes, the uptake of the molecule is very specific and is controlled by recognition of the molecule by a specific receptor on the surface of the cell. This process is called Receptor Mediated Endocytosis. Refer to page.
ADP/ATP Exchange This tutorial explains ADP/ATP exchange, a form of Antiport transport, a mechanism where two different molecules facilitate each others pass through the membrane in opposite directions. Refer to page.
DNA Replication The enzymatic and structural features of DNA replication in all organisms are very similar. These animations illustrate the processes involved in the replication of the Eschericia coli chromosome. The names of the DNA sites, enzymes, and other protein factors differ in viruses and in eukaryotic organisms, however, the basic features have been highly conserved in evolution. In E. coli, DNA replication begins at a single site on the chromosome called "OriC". Refer to page.
DNA Transcription This tutorial shows the steps involved in transcribing DNA into mRNA. Transcription is illustrated using the E. coli lactose operon (lac) where transcription is regulated negatively by the lac repressor and positively by CAP-cAMP complex. Refer to page.
RNA Translation - Protein Synthesis The biosynthesis of proteins involves translation of the information contained in the mRNA into a polypeptide sequence. Each triplet of bases in the mRNA encodes one amino acid. The translation is accomplished by the ribosome with the use of specialized RNA molecules called transfer RNA, or tRNA. The tRNA molecules bind to the mRNA and deliver the correct amino acid to the growing polypeptide chain. Refer to page.
ATP Synthesis The synthesis of ATP utilizing the proton gradient as a source of energy is an example of direct coupling. The energy released as the protons flow through the enzyme cause a conformational change in the protein that causes the formation of ATP from bound ADP and inorganic phosphate. Refer to page.
Serine Protease The tutorial shows the chemical mechanism of serine proteases, enzymes that in the family differ only in their substrate specificity.The Trypsin for example is an extracellular protease that hydrolyzes peptide bonds during digestion in the small intestine.
Metabolism Overview The four key pathways, glycolysis, the TCA cycle, electron transport, and ATP synthesis are outlined in yellow. Glucose that is brought into the cell via the glucose transporter can suffer two fates, oxidation or storage as glycogen. Oxidation occurs in glycolysis and the TCA cycle, releasing the carbon atoms in glucose as CO2. Note that oxygen is not used until the end of the electron transport chain. High energy electrons, symbolized as orange balls are carried on organic electron carriers to the electron transport chain. As these electrons move through the four complexes, protons are pumped from the mitochondrial matrix across the inner mitochondrial membrane. As these protons flow back through the membrane via ATP synthase, ATP is generated. Refer to page.

The following is the list of simulations provided within the various modules of the course.

Dissociation of Weak Electrolytes Some molecules are weak electrolytes and exist in a reversible equilibrium between the starting molecule and its dissociated parts. For molecules that are weak electrolytes and act as acids (proton donors), the ratio of the products of the dissociated parts and the parent molecule is a constant (K) in neutral water for each separate molecular structure. Refer to page.
Equilibrium of MO complex formation Equilibrium state as determined by protein and ligand concentrations: This simulation allows you to explore the equilibrium between free protein, ligand, and the complex, and how ligand concentration affects the equilibrium. Refer to page.
Enzyme Catalysis Enzymes bind to substrates in a manner similar to the way myoglobin binds oxygen or the estradiol binds to the estrogen receptor, but enzymes can go one step further. In this case the ligand is specifically referred to as the substrate (the molecule that the enzyme will convert to product) and it binds to a specific binding region of the enzyme referred to as the active site. Once bound, the ligand, or substrate, can either simply reversibly come off the enzyme, or it can be converted into a new compound or product. Refer to page.
Membrane Permeability The introduction of a cell or liposome to the solution places a barrier to the molecules. As three different molecules diffuse to equilibrium in the following simulation, they encounter the lipid bilayer depicted by the horizontal membrane across the center of the stage. Note that one type of molecule passes freely through the lipid bilayer while the second type of molecule only occasionally passes through the membrane and the lipid bilayer is totally impermeable to the third type of molecule. These molecules move through the membranes via passive diffusion. Refer to page.
Osmosis: Isotonic Equilibrium Cells continually encounter changes in their external ionic environment and will spontaneously respond by attempting to equalize the concentration of ions on the inside and outside of the cell. Because the plasma membrane (lipid bilayer) is significantly less permeable to ions than water, the establishment of an equal concentration of the ions on either side of the membrane is accomplished by the net movement of water toward the higher concentration of ions to reduce the concentration. This movement of water in response to an imbalance of solute (ion) is referred to as osmosis. Refer to page.
Intercellular Transport: Gap Junctions This simulation demonstrates how transmembrane protein structures from adjacent cells line up to form Gap Junctions, channels between cells that act as size exclusion transporters. While water molecules are small enough to move through the membranes, the Gap Junctions facilitate that movement and the movement of molecules up to 1500 daltons (approximately a 15 amino acid peptide) but not larger molecules. Refer to page.
Product inhibition This simulation demonstrates how a compound that is further down the pathway, or even a compound in a separate pathway, can inhibit a reaction Refer to page.
Allosteric Binding This simulation demonstrates how Allosteric binding causes conformational changes to an enzyme that can inhibit or activate Enzyme-substrate binding. Refer to page.
Calculating Free Energy This simulation demonstrates how to calculate the Gibbs free energy in a controlled reaction. Refer to page.

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Use this table as a quick reference for the major elements.

Atomic Properties of the major biological atoms
Atom Mass Covalent Bonds Electronegativity
H 1 1 2.20
C 12 4 2.55
N 14 3 or 4 (e.g. NH4+) 3.04
O 16 2 3.44
P 31 5 2.10
S 32 2 2.58