3 The Control of Enzyme Mediated Reactions and Bioenergetics PDF

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The Control of Enzyme-Mediated Reactions and Bioenergetics

3.1

Regulation of Enzyme-Mediated Reactions

The rate of an enzyme-catalyzed reaction depends on numerous factors, including the concentration of the enzyme and the pH and temperature of the solution. Genetic control of enzyme concentration, for example, affects the rate of progress along particular metabolic pathways and thus regulates cellular metabolism. The activity of an enzyme, as measured by the rate at which its substrates are converted to products, is influenced by such factors as: (1)

The temperature and pH of the solution;

(2) The concentration of cofactors and coenzymes, which are needed by many enzymes as “helpers” for their catalytic activity; (3) The concentration of enzyme and substrate molecules in the solution; and (4) The stimulatory and inhibitory effects of some products of enzyme action on the activity of the enzymes that helped to form these products.

3.1.1

The Effects of Temperature and pH

An increase in temperature will increase the rate of nonenzyme-catalyzed reactions. A similar relationship between temperature and reaction rate occurs in enzyme-catalyzed reactions (Figure 3.1).

Figure 3.1: The effect of temperature on enzyme activity. This effect is measured by the rate of the enzyme-catalyzed reaction under standardized conditions as the temperature of the reaction is varied. 1

At a temperature of 0°C the reaction rate is immeasurably slow. As the temperature is raised above 0°C the reaction rate increases, but only up to a point. At a few degrees above body temperature (which is 37°C) the reaction rate reaches a plateau; further increases in temperature actually decrease the rate of the reaction (Figure 3.1). This decrease is due to the fact that the tertiary structure of enzymes becomes altered at higher temperatures. A similar relationship is observed when the rate of an enzymatic reaction is measured at different pH values. Each enzyme characteristically exhibits peak activity in a very narrow pH range, which is the pH optimum for the enzyme. If the pH is changed so that it is no longer within the enzyme’s optimum range, the reaction rate will decrease (Figure 3.2). This decreased enzyme activity is due to changes in the conformation of the enzyme and in the charges of the R groups of the amino acids lining the active sites.

Figure 3.2: The effect of pH on the activity of three digestive enzymes. Salivary amylase is found in saliva, which has a pH close to neutral; pepsin is found in acidic gastric juice, and trypsin is found in alkaline pancreatic juice. The pH optimum of an enzyme usually reflects the pH of the body fluid in which the enzyme is found. The acidic pH optimum of the protein-digesting enzyme pepsin, for example, allows it to be active in the strong hydrochloric acid of gastric juice. Similarly, the neutral pH optimum of salivary amylase and the alkaline pH optimum of trypsin in 2

pancreatic juice allow these enzymes to digest starch and protein, respectively, in other parts of the digestive tract.

3.1.2 Cofactors and Coenzymes Many enzymes are completely inactive when isolated in a pure state. Evidently some of the ions and smaller organic molecules that are removed in the purification procedure play an essential role in enzyme activity. These ions and smaller organic molecules needed for the activity of specific enzymes are called cofactors and coenzymes.

3.1.2.1 Cofactors Cofactors include metal ions such as Ca2+, Mg2+ , Mn2+, Cu2+, Zn2+ , and selenium. Some enzymes with a cofactor requirement do not have a properly shaped active site in the absence of the cofactor. In these enzymes, the attachment of cofactors causes a conformational change in the protein that allows it to combine with its substrate (this is a form of allosteric modulation). The cofactors of other enzymes participate in the temporary bonds between the enzyme and its substrate when the enzyme-substrate complex is formed (Figure 3.3). Since only a few enzyme molecules need be present to catalyze the conversion of large amounts of substrate to product, very small quantities of these trace metals are sufficient to maintain enzymatic activity.

Figure 3.3: The roles of cofactors in enzyme function. In (a) the cofactor changes the conformation of the active site, allowing for a better fit between the enzyme and its substrates. In (b) the cofactor participates in the temporary bonding between the active site and the substrates.

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3.1.2.2 Coenzymes Coenzymes are organic molecules, derived from water-soluble vitamins such as niacin and riboflavin that are needed for the function of particular enzymes. Coenzymes participate in enzyme-catalyzed reactions by transporting hydrogen atoms and small molecules from one enzyme to another. Enzymes that require coenzymes catalyze reactions in which a few atoms (for example, hydrogen, acetyl, or methyl groups) are either removed from or added to a substrate. For example:

What makes a coenzyme different from an ordinary substrate is the fate of the coenzyme. In our example, the two hydrogen atoms that are transferred to the coenzyme can then be transferred from the coenzyme to another substrate with the aid of a second enzyme. This second reaction converts the coenzyme back to its original form so that it becomes available to accept two more hydrogen atoms (Figure 3.4). A single coenzyme molecule can be used over and over again to transfer molecular fragments from one reaction to another. Thus, as with metallic cofactors, only small quantities of coenzymes are necessary to maintain the enzymatic reactions in which they participate.

Figure 9.10: The coenzymes nicotinamide adenine dinucleotide (NAD+) and flavine adenine dinucleotide (FAD) are used to transfer two hydrogen atoms from one reaction 4

to a second reaction. In the process, the hydrogen-free forms of the coenzymes are regenerated. Coenzymes are derived from several members of a special class of nutrients known as vitamins. For example, the coenzymes NAD+ (nicotinamide adenine dinucleotide) and FAD (flavine adenine dinucleotide) (Figure 3.4) are derived from the B-vitamins niacin and riboflavin, respectively. As we shall see, they play major roles in energy metabolism by transferring hydrogen from one substrate to another.

3.1.3 The Effect of Concentration of Enzyme and Substrate Molecules 3.1.3.1 Enzyme Concentration At any substrate concentration, including saturating concentrations, the rate of an enzyme-mediated reaction can be increased by increasing the enzyme concentration. In most metabolic reactions, the substrate concentration is much greater than the concentration of enzyme available to catalyze the reaction. Therefore, if the number of enzyme molecules is doubled, twice as many active sites will be available to bind substrate, and twice as many substrate molecules will be converted to product (Figure 3.5). Certain reactions proceed faster in some cells than in others because more enzyme molecules are present.

Figure 3.5: Rate of an enzyme-catalyzed reaction as a function of substrate concentration at two enzyme concentrations, A and 2A. Enzyme concentration 2A is twice the enzyme concentration of A, resulting in a reaction that proceeds twice as fast at any substrate concentration. 5

In order to change the concentration of an enzyme, either the rate of enzyme synthesis or the rate of enzyme breakdown must be altered. Since enzymes are proteins, this involves changing the rates of protein synthesis or breakdown. Regardless of whether altered synthesis or altered breakdown is involved, changing the concentration of enzymes is a relatively slow process, generally requiring several hours to produce noticeable changes in reaction rates.

3.1.3.2 Substrate Concentration and Reversible Reactions At a given level of enzyme concentration, the rate of product formation will increase as the substrate concentration increases. Eventually, however, a point will be reached where additional increases in substrate concentration do not result in comparable increases in reaction rate. When the relationship between substrate concentration and reaction rate reaches a plateau of maximum velocity, the enzyme is said to be saturated. If we think of enzymes as workers in a plant that converts a raw material (say, metal ore) into a product (say, iron), then enzyme saturation is like the plant working at full capacity, with no idle time for the workers. Increasing the amount of raw material (substrate) at this point cannot increase the rate of product formation. This concept is illustrated in Figure 3.6.

Figure 3.6: The effect of substrate concentration on the rate of an enzyme-catalyzed reaction. When the reaction rate is at a maximum, the enzyme is said to be saturated.

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Some enzymatic reactions within a cell are reversible, with both the forward and backward reactions catalyzed by the same enzyme. The enzyme carbonic anhydrase, for example, is named because it can catalyze the following reaction:

The same enzyme, however, can also catalyze the reverse reaction:

The two reactions can be more conveniently illustrated by a single equation with double arrows:

The direction of the reversible reaction depends, in part, on the relative concentrations of the molecules to the left and right of the arrows. If the concentration of CO2 is very high (as it is in the tissues), the reaction will be driven to the right. If the concentration of CO2 is low and that of H2CO3 (carbonic acid) is high (as it is in the lungs), the reaction will be driven to the left. The principle that reversible reactions will be driven from the side of the equation where the concentration is higher to the side where the concentration is lower is known as the law of mass action. Although some enzymatic reactions are not directly reversible, the net effects of the reactions can be reversed by the action of different enzymes. Some of the enzymes that convert glucose to pyruvic acid, for example, are different from those that reverse the pathway and produce glucose from pyruvic acid. Likewise, the formation and breakdown of glycogen (a polymer of glucose) are catalyzed by different enzymes.

3.1.4 Enzyme Activation and Inhibition 3.1.4.1 Activation and Inhibition of Enzymes There are a number of important cases in which enzymes are produced as inactive forms. In the cells of the pancreas, for example, many digestive enzymes are produced 7

as inactive zymogens, which are activated after they are secreted into the intestine. Activation of zymogens in the intestinal lumen (cavity) protects the pancreatic cells from self-digestion. In liver cells, as another example, the enzyme that catalyzes the hydrolysis of stored glycogen is inactive when it is produced, and must later be activated by the addition of a phosphate group. A different enzyme, called a protein kinase, catalyzes the addition of the phosphate group to that enzyme. At a later time, enzyme inactivation is achieved by another

enzyme

that

catalyzes

the

removal

of

the

phosphate

group.

The

activation/inactivation of this enzyme (and many others) is thus achieved by the processes of phosphorylation/dephosphorylation. Going back a step, the protein kinase itself may be produced as an inactive enzyme. In this case, activation of the protein kinase requires that it bind to a particular ligand (smaller molecule) (Figure 3.7). Such ligands serve as intracellular regulators that are called second messengers. In many cases, this ligand is a molecule called cyclic AMP (cAMP). Cyclic AMP activates the protein kinase by promoting the dissociation of an inhibitory subunit from the active enzyme. The production of cyclic AMP within cells is stimulated by regulatory molecules that include neurotransmitters and hormones.

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Figure 3.7: Activation and inactivation mechanisms of protein kinase A (PKA). The PKA enzyme is also known as cAMP-dependent enzyme because it gets activated only if cAMP is present. (A) Activation of protein kinase A of higher eukaryotes via cAMP. The freed C-subunits are phosphorylating various substrate proteins at specific serine or threonine sites, respectively. Hereby ATP is converted to ADP. (B) One of the substrates of PKA is the phosphodiesterase (PDE). After phosphorylation by PKA, the activated enzyme catalyzes the hydrolysis of cAMP to AMP. Hence the activity of PKA is reduced or terminated (feedback control).

3.1.5 Section Summary: Mediated Reactions

Regulation

of

Enzyme-

1. The activity of an enzyme is affected by a variety of factors. 2. The rate of enzyme-catalyzed reactions increases with increasing temperature, up to a maximum. (1) This is because increasing the temperature increases the energy in the total population of reactant molecules, thus increasing the proportion of reactants that have the activation energy. (2) At a few degrees above body temperature, however, most enzymes start to denature, which decreases the rate of the reactions that they catalyze. 9

3. Each enzyme has optimal activity at a characteristic pH—called the pH optimum for that enzyme. (1) Deviations from the pH optimum will decrease the reaction rate because the pH affects the shape of the enzyme and charges within the active site. (2) The pH optima of different enzymes can vary widely—pepsin has a pH optimum of 2, for example, while trypsin is most active at a pH of 9. 4. Many enzymes require metal ions in order to be active. These ions are therefore said to be cofactors for the enzymes. 5. Many enzymes require smaller organic molecules for activity. These smaller organic molecules are called coenzymes. (1) Coenzymes are derived from water-soluble vitamins. (2) Coenzymes transport hydrogen atoms and small substrate molecules from one enzyme to another. 6. Some enzymes are produced as inactive forms that are later activated within the cell. (1) Activation may be achieved by phosphorylation of the enzyme, in which case the enzyme can later be inactivated by dephosphorylation. (2) Phosphorylation of enzymes is catalyzed by an enzyme called protein kinase. (3) Protein kinase itself may be inactive and require the binding of a second messenger called cyclic AMP in order to become activated. 7. The rate of enzymatic reactions increases when either the substrate concentration or the enzyme concentration is increased. (1) If the enzyme concentration remains constant, the rate of the reaction increases as the substrate concentration is raised, up to a maximum rate. (2) When the rate of the reaction does not increase upon further addition of substrate, the enzyme is said to be saturated.

3.2

Metabolic Pathways

3.2.1 The General Pattern of a Metabolic Pathway The many thousands of different types of enzymatic reactions within a cell do not occur independently of each other. They are, rather, all linked together by intricate webs of interrelationships, the total pattern of which constitutes cellular metabolism. A sequence of enzymatic reactions that begins with an initial substrate, progresses

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through a number of intermediates, and ends with a final product is known as a metabolic pathway. The enzymes in a metabolic pathway cooperate in a manner analogous to workers on an assembly line, where each contributes a small part to the final product. In this process, the product of one enzyme in the line becomes the substrate of the next enzyme, and so on (Figure 3.8).

Figure 3.8: The general pattern of a metabolic pathway. In metabolic pathways, the product of one enzyme becomes the substrate of the next. Few metabolic pathways are completely linear. Most are branched so that one intermediate at the branch point can serve as a substrate for two different enzymes. Two different products can thus be formed that serve as intermediates of two pathways (Figure 3.9).

Figure 3.9: A branched metabolic pathway. Two or more different enzymes can work on the same substrate at the branch point of the pathway, catalyzing two or more different reactions.

3.2.2 End-Product Inhibition 11

The activities of enzymes at the branch points of metabolic pathways are often regulated by a process called end-product inhibition, which is a form of negative feedback inhibition. In this process, one of the final products of a divergent pathway inhibits the activity of the branch-point enzyme that began the path toward the production of this inhibitor. This inhibition prevents that final product from accumulating excessively and results in a shift toward the final product of the alternate pathway (Figure 3.10). The mechanism by which a final product inhibits an earlier enzymatic step in its pathway is known as allosteric inhibition. The allosteric inhibitor combines with a part of the enzyme at a location other than the active site. This causes the active site to change shape so that it can no longer combine properly with its substrate.

Figure 3.10: End-product inhibition in a branched metabolic pathway. Inhibition is shown by the arrow in step 2.

3.2.3 Inborn Errors of Metabolism Since each different polypeptide in the body is coded by a different gene, each enzyme protein that participates in a metabolic pathway is coded by a different gene. An inherited defect in one of these genes may result in a disease known as an inborn error of metabolism. In this type of disease, the quantity of intermediates formed prior to the defective enzymatic step increases, and the quantity of intermediates and final products formed after the defective step decreases. Diseases may result from deficiencies of the normal end product or from excessive accumulation of intermediates 12

formed prior to the defective step. If the defective enzyme is active at a step that follows a branch point in a pathway, the intermediates and final products of the alternate pathway will increase (Figure 3.11). An abnormal increase in the production of these products can be the cause of some metabolic diseases.

Figure 3.11: The effects of an inborn error of metabolism on a branched metabolic pathway. The defective gene produces a defective enzyme, indicated here by a line through its symbol. One of the conversion products of phenylalanine is a molecule called DOPA, an acronym for dihydroxyphenylalanine. DOPA is a precursor of the pigment molecule melanin, which gives skin, eyes, and hair their normal coloration. The condition of albinism results from an inherited defect in the enzyme that catalyzes the formation of melanin from DOPA (Figure 3.12). Besides PKU and albinism, there are many other inborn errors of amino acid metabolism, as well as errors in carbohydrate and lipid metabolism. Some of these are described in Table 3.1.

Figure 3.12: Metabolic pathways for the degradation of the amino acid phenylalanine. Defective enzyme1 produces phenylketonuria (PKU), defective enzyme5 produces 13

alcaptonuria (not a clinically significant condition), and defective enzyme6 produces albinism. Table 3.1: Examples of inborn errors in the metabolism of amino acids, carbohydrates, and lipids

3.2.4 Enzyme Activity 3.2.4.1 What Does Enzyme Activity Mean? Enzyme activity is a measure of the ability of a given enzyme to convert its substrate(s) into its product(s). Depending on the enzyme it is typically assayed by measuring either the amount of substrate that's disappearing, or the amount of product that's appearing over a specified period of time, such that the final result is expressed in terms of moles of conversion per unit mass of protein per unit time, for example, nmol/mg protein/sec. Officially, enzyme activity is defined in terms of "U...