Enzymes I, II, and III The Role of Enzymes A catalyst increases the rate or velocity of a chemical reaction without

itself being changed in the overall process. Most biological catalysts are proteins called enzymes. The substance acted on by an enzyme is called asubstrate. Enzymes speed up reactions by many orders of magnitude. For example, the enzyme catalase speeds up the conversion of hydrogen peroxide to water and oxygen by a factor of a billion. True catalysts, such as enzymes, participate in the reaction, but are unchanged by it. Therefore, they can continue to catalyze subsequent reactions. Catalysts change the rates of reactions, but do not affect the equilibrium of a reaction. That is, you cannot make more product from an enzyme-catalyzed reaction than you can from the same reaction without it. The enzyme simply helps to reach the equilibrium state faster than if it were not present. Classification of Protein Enzymes To reduce confusion in the naming of enzymes, the Enzyme Commission (EC) of the International Union of Biochemistry and Molecular Biology (IUBMB) devised a naming and numbering system. In it, enzymes are divided into six major classes, with sub-groups and subsubgroups to define their functions more precisely. The major classes are as follows (Table 11.7): 1. 2. 3. 4. Oxidoreductases catalyze oxidation - reduction reactions. Transferases catalyze transfer of functional groups from one molecule to another. Hydrolases catalyze hydrolytic cleavage. Lyases catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement. 5. Isomerases catalyze intramolecular rearrangement. 6. Ligases catalyze reactions in which two molecules are joined. The EC of the IUBMB has given each enzyme a number with four parts, such as EC 3.4.21.5. The first three numbers define major class, subclass, and sub-subclass, respectively. The last is a serial number in the sub-subclass, indicating the order in which each enzyme is added to the list, which is continually growing. For example, triose phosphate isomerase is listed as EC 5.3.1.1. Thus, it is an isomerase and in the third subclass (enzymes that involve an oxidation in one part of the substrate molecule and reduction in another). It is in the first sub-subclass (those that interconvert aldoses and ketoses) and is the first entry (of 19 so far) in this sub-subclass. How Catalysts Work A catalyst works simply by lowering the energy barrier of a reaction, (Figure 11.2, Figure 11.4). By doing so, the catalyst increases the fraction of molecules that have enough energy to attain the transition state, thus making the reaction go faster in both directions. The position of the equilibrium (the amount of product versus reactant) is unchanged by a catalyst. Even though k1 and k-1 many be greatly changed from their values in the absence of a catalyst,

each one changes by the same factor and the equilibrium constant, K, is unchanged, because K = k1/k-1. Catalysts lower the energy barrier in two ways: 1. The catalyst binds a substrate in an intermediate conformation that resembles the transition state, but has a lower energy. This may lead to multiple intermediate states that bypass the transition state (Figure 11.5 see book - Figure misnumbered). An intermediate state is a metastable state of a molecule. 2. In a non-catalyzed reaction the entropy may be highly negative due to the highly specific orientation required in order for a reaction to occur. (Note that a more negative entropy contributes to a more positive free energy of transition.) Catalysts can lower the negative entropy by binding reacting molecules only in the proper mutual orientataion, thus increasing their reactivity (Figure 11.6). The Induced Fit Model The induced fit model of enzyme action is a modification of the lock-and-key model originally proposed by Emil Fischer in 1894 (Figure 11.7a). The lock-and-key model proposes that an enzyme/substrate pair is like a lock and key. Though it explains the specificity of enzyme/substrate pairs, it does little to explain catalysis, because a lock does not change a key the way an enzyme changes a substrate. In 1958, Daniel Koshland proposed the induced fit model to explain enzymatic catalysis (Figure 11.7b). The model proposes that distortion of the enzyme and the substrate is an important event in catalysis. Figure 11.8shows x-ray diffraction studies of the enzyme hexokinase both without (11.8a) and with (11.8b) glucose bound. Note that binding of glucose causes two domains of the enzyme to fold toward each other. Enzymes do more than simply bind and position substrates, however. Enzymes 1. Bind substrate(s); 2. Lower the energy of the transition state; and 3. Directly promote the catalytic event. The latter may occur as a result of specific amino acid side chains that physically interact with the substrate and end up promoting the reaction. When the catalytic process is complete, the enzyme must be able to release the product or products and return to its original state for another round of catalysis. For an enzyme (E) that catalyzes the conversion of a single substrate (S) into a single product (P), the simplest way to write the overall reaction is in two steps: S + E <=> ES ES -> E + P

Here, ES represents the enzyme-substrate complex. For simplicity, the first reaction is shown as reversible, while the second reaction is irreversible. For specific examples, see the triose phosphate isomerase and serine proteases in your book. Function of Coenzymes Some kinds of biological processes require catalytic functions beyond those built into protein molecules alone. In such cases, a protein may require the help of some other small molecule or ion to carry out the reaction. Molecules bound to enzymes for this purpose are called coenzymes. The water soluble vitamin B complexes are metabolic precursors of a number of coenzymes. Table 11.5 lists several important coenzymes together with their related vitamins. NAD+ - Nicotinamide adenine dinucleotide (NAD+) is derived from the vitamin niacin. The nicotinamide portion of the molecule is capable of being reduced and can thus serve as an oxidizing agent (seehere), where 'R' stands for the remainder of the molecule. NAD+/NADH behave both like a second substrate in a reaction (because each is converted to the other by the enzyme) and like a coenzyme (because they are recycled over and over). They are generally classified as coenzymes. Metal Ions in Coenzymes - Many enzymes contain metal ions, usually held by coordinate covalent bonds from amino acid side chains, but sometimes bound by a prosthetic group like heme. Such enzymes are calledmetalloenzymes. Figure 11.27 shows the active site of the protease carboxypeptidase A, which contains a zinc ion. Covalent Modifications to Regulate Enzyme Activity Covalent modification activates some enzymes and inactivates others. That is, some enzymes are wholly inactive until they are covalently modified and then begin to function. In other cases, covalent modification acts in the opposite direction, to inactivate otherwise active enzymes. Some such modifications can be reversed; others cannot. One of the most widespread modifications is phosphorylation or dephosphorylation of various amino acid side chains (e.g., serine, threonine, tyrosine, and histidine). These kinds of modification are most often a part of complex regulatory pathways, frequently under hormonal control. (See kinase cascade). Another example of covalent enzyme activation is proteolytic cleavage, found in the pancreatic proteases (such as trypsin, chymotrypsin, elastase, and carboxypeptidase). These enzymes are synthesized in the pancreas in an inactive form because if they were active in the pancreas, they would digest the pancreatic tissue. Rather, they are made as slightly longer, catalytically inactive molecules called zymogens (trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase, respectively). The zymogens must be cleaved proteolytically in the intestine to yield the active enzymes (Figure 11.39). If a small amount of protease becomes active in the pancreas, it can have painful or fatal consequences (i.e., acute pancreatitis). The pancreas protects itself from active protease action by synthesis of a protein called the secretory pancreatic trypsin inhibitor. The binding between trypsin and its inhibitor is one of the strongest noncovalent interactions known in biochemistry. The intestinal tissue is protected somewhat from damage by proteases by its glycosylated surface.

The first step is activation of trypsin in the duodenum. A hexapeptide is removed from the Nterminal end of trypsinogen by enteropeptidase,a protease secreted by duodenal cells. This yields active trypsin, which then activates the other zymogens by specific proteolytic cleavages. Trypsin will also activate other trypsinogen molecules in an autocatalytic process. Activation of a few trypsinogens ultimately leads to activation of many trypsins. Activation of chymotrypsinogen is shown in Figure 11.40. First, trypsin cleaves the bond between arginine 15 and isoleucine 16. Notice that the N-terminal peptide remains attached to the rest of the molecule due to the disulfide bond between residues 1 and 122. The enzyme is activated by the cleavage due to changes in the conformation of the molecule. These include: Creation of a new, positively charged N-terminal residue at Ile 16; Salt bridge formation between Ile 16 and Asp 194 (next to the active site); and Movement of active site residues so that the amino groups of residues 193 and 195 are properly positioned to hydrogen-bond to the substrate oxyanion in the tetrahedral transition state. Finally, autocatalytic cleavages to remove residues 14-15 and 147-148 from the molecule produce the final, active form of chymotrypsinogen, called -chymotrypsin. Blood Clotting Blood clots are composed of striated fibers of a protein called fibrin. Fibrin fibers stick together in a staggered array. Before fibrin fibers can stick together, however, they must be derived from a zymogen precursor, called fibrinogen, by proteolytic cleavages that release fibrinopeptides A and B from the molecule. Removal of these small fibrinopeptides exposes sites in the fibrin molecules that allows them to stick together. Covalent cross-links between glutamine and lysine residues also form to help stabilize the structure. Thus, activation of zymogens is a key aspect to clotting of blood in vertebrates. As can be seen in Figure 11.42, production of fibrin is the product of action of a cascade of proteases. These are summarized as follows:

In damaged tissue, the proteins kininogen and kallikrein activate factor XII (part of the intrinsic pathway). Factor XII activates factor XI (part of the intrinsic pathway). Alternatively, damage to blood vessels leads to release of tissue factor and activation of factor VII (start of the extrinsic pathway). The extrinsic and intrinsic pathways merge with activation of factor X. Activation of factor X by factor IX of the intrinsic pathway requires factor VIII (antihemophilic factor). Factor X activates prothrombin to thrombin. Thrombin removes small fibrinopeptides from fibrinogen to form fibrin.

Note that the absence of factor VIII is the cause of classic hemophilia. Factor VIII is encoded on the X chromosome, so the disease is much more prominent in males, because they have only one X chromosome. As wounds heal, clots must be removed. The principal agent for dissolving clots is an enzyme called plasmin, which is synthesized as the inactive zymogen called plasminogen. Plasminogen

is activated by a number of proteases, the most important of which is tissue-type plasminogen activator (t-PA). t-PA can be extremely effective in initiating the cascade to dissolve the unwanted blood clot involved in stroke or heart attack. Molecular Engineering Molecular engineering is a term loosely used to describe the design of enzymes using modern molecular biological techniques to alter their catalytic action. Examples include the following: Site-directed mutagenesis - In this technique, the DNA coding sequence for an enzyme is altered to change one or more amino acids in an enzyme when the mutated DNA is expressed in an organism. Hybrid enzymes - Here, molecular techniques allow researchers to put together two different biomolecules to make a fusion molecule with new, useful properties. Figure 11.28 depicts a hybrid enzyme made in this fashion. In this case, an oligonucleotide of a defined sequence has been grafted onto the enzyme staphylococcal nuclease. The specific sequence in the hybrid enzyme allows it to bind to a specific complementary nucleic acid sequence (specified by the bound oligonucleotide) and cut specifically at that point. The native, unaltered enzyme has no such specificity. Catalytic antibodies - These interesting molecules are antibodies with a very specific binding site to the transition state of an enzymatic reaction. The resulting molecules, called abzymes, act like antibodies. In some cases, abzymes can speed up reaction rates as much as 10 7-fold over the uncatalyzed reaction. The stereospecificity of enzymes (including abzymes) may provide a tremendous aid to the synthesis of stereospecific compounds in organic chemistry. Ribozymes In most cases, proteins called enzymes (or abzymes-see above) catalyze chemical reactions. It turns out, however, that some RNA molecules, called ribozymes are capable of catalyzing chemical reactions too. Figure 11.29 shows the site of action of the RNA-protein complex called ribonuclease P. The RNA portion of the complex can, by itself, catalyze the hydrolysis of the specific bond indicated by the red wedge in the figure. Tom Cech identified an interesting protein-independent self-splicing agent from the preribosomal RNA of the protist, Tetrahymena. In this reaction, the rRNA itself catalyzes removal of an RNA intron from itself. The RNA molecule involved in the catalysis is altered, so it is not technically considered a catalyst, but the sequence which is removed (called L-19 IVS) does have true catalytic activity. It can either lengthen or shorten small oligonucleotides, in the manner shown in Figure 11.30. RNAs can catalyze reactions in which evolution of more efficient molecules based on selection can occur. Such processes may have been of importance in a pre-protein world during the origin of life. Regulation of Enzyme Activity Enzymes function in assembly line-like fashion to catalyze the thousands of reactions occurring in cells each second. Coordinating and regulating enzymatic activities is essential for efficient

functioning of cells. Several control mechanisms that do not involve covalent modification of the enzymes are possible: Substrate level control - In this control mechanism, high levels of the product of a reaction inhibit the ability of the small amounts of substrate present to react. An example is the first step in glycolysis, catalyzed by hexokinase. It is inhibited by the product of its action, glucose-6phosphate. If glycolysis is blocked for any reason, glucose-6-phosphate accumulates. Feedback control - In this mechanism, the product of a series of reactions (like in an assembly line) inhibits the action of an earlier step in the process (usually the first step). Feedforward regulation occurs when a molecule in an assembly line reaction activates an enzyme ahead of it in the pathway. Allosteric enzymes - These enzymes are invariably multisubunit proteins, with multiple active sites. They exhibit cooperativity in substrate binding (homoallostery) and regulation of their activity by other, effector molecules (heteroallostery). Homoallostery - The effects of cooperative substrate binding on enzyme kinetics are shown in Figure 11.32. Binding of one substrate favors binding of additional substrates. Cooperative binding favors reduction of KM for the binding of substrates after the initial one. Figure 11.33 shows the effect of extreme homoallostery. At concentrations of S below a critical point, [S]c, the enzyme is almost inactive, but then changes activity rapidly with concentrations of S greater than [S]c. Heteroallostery - This type of allosteric control involves heteroallosteric effectors which may be either inhibitors or activators of binding. If an enzyme can exist in two conformational states, T and R, that differ dramatically in the strength with which substrate is bound or which differ significantly in the catalytic rate, then the kinetics of the enzyme can be controlled by any other substance that, in binding to the protein, shifts the T<=>R equilibrium. Allosteric inhibitors shift the equilibrium toward T and activators shift it toward the R state. Figure 11.34 illustrates how heteroallosteric control of an enzyme affects the shape of a V-vs-[S] curve. Note that shifts toward the R state (activators) increase the velocity for a given substrate concentration, whereas shifts toward the T state have the opposite effect. Aspartate Carbamoyltransferase Aspartate carbamoyltransferase (also known as aspartate transcarbamoylase or ATCase) is one of the most studied enzymes for allosteric regulation. It catalyzes the reaction that follows: Aspartate + Carbamoyl Phosphate <=> Carbamoyl Aspartate As seen in Figure 11.35, ATCase is at the crossroads of biosynthetic pathways that lead to proteins or pyrimidines. ATCase catalyzes the joining of aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate, the first metabolite committed to the synthesis of pyrimidines. The enzyme is sensitive to feedback inhibition by cytidine triphosphate ( CTP), one of the end products of the pathway, and is activated by ATP, a purine nucleotide (Figure 11.36). ATCase is a multisubunit protein consisting of 6 catalytic subunits and 6 regulatory subunits.The quarternary structure of ATCase enzyme is shown in Figure 11.37. A detailed representation of one catalytic subunit and one regulatory subunit is shown in Figure 11.38.

Allosteric regulation of ATCase involves changes in the quaternary structure of the molecule. A major rearrangement of subunit positions occurs in the T-> R transition. Sometimes different organisms regulate similar pathways in different ways. For example, ATCase is the major control point in the pyrimidine pathway synthesis in bacteria, whereas eukaryotes regulate the synthesis of carbamoyl phosphate ( Figure 11.35). In mammals, the carbamoyl phosphate synthetase II is inhibited by UDP, UTP, CTP, dUDP, and UDPglucose. Reaction Order First order reactions - For the irreversible reaction, A -> B, the reaction rate (velocity, V) is given by V = d[B]/dT for the rate of appearance of the product, B, or by V = -d[A]/dT for the rate of disappearance of the substrate, A. These equations are equally valid for this reaction, so we can say V = d[B]/dT = -d[A]/dT = k1[A], where k1 is called the rate constant and for this reaction has units of (seconds) -1. This type of a reaction is called a first-order reaction, because its rate depends on the first power of the reactant concentration. If k1 is large, the reaction is fast and if k1 is small, the reaction is slow. Integrating the above equation yields [A]/[A]0 = e-(k-1t), where [A]0 is the starting concentration of A when t = 0. A plot of this equation (Figure 11.1a) shows that the concentration of A decreases exponentially with time. The amount of time it takes for half of A to be lost is called the half-life and is given by t1/2. The half-life is inversely proportional to k1. A plot of ln[A] vs t, as shown in Figure 11.1b, will always yield a straight line with slope = -k1 if the reaction is first order. If one plots the initial rate of the reaction versus varying starting concentrations ([A]0), a straight line plot with slope of k1 will be produced for a first order reaction. The most common example of a first order reaction is the decay of radioactive elements. This approach to reactions in biological cells is too simple to explain them properly. One reason is that many of the reactions and processes in cells are reversible-that is, the equilibrium does not lie far to one side and, as product accumulates, the reverse reaction becomes important.

So, for the reaction A < = > B, a forward reaction constant, k1, can be used to define the reaction moving rightward and a reverse reaction constant, k--1 can be used to define the reaction moving leftward. Now the molecule A is being consumed in the reaction to the right and formed by the reaction to the left, so the corresponding rate equation is -V = d[A]/dt = -k1[A] + k-1[B] Here, k1 and k-1 are the rate constants for the first-order forward and reverse reactions. Such a reaction approaches a state of equilbrium, at which point the rates of the forward and reverse reactions become equal. At the same time, the overall rate becomes zero, so 0 = -k1[A]eq + k-1[B]eq or [B]eq/[A]eq = k1/k-1 = K, where K is the equilibrium constant. For a reversible reaction that is first-order in both directions, the equilibrium constant is always the ratio of the forward and reverse rate constants. Second-Order Reactions - A reaction of this type typically occurs when two molecules come together to form products. A simple example is 2A -> A2 with a rate constant given by k2. The rate of such a reaction is proportional to the second power of the concentration of the reactant. Therefore, V = -d[A]/dt = -k2[A]2 Here k2 is the second-order rate constant. It has dimensions of (mol/L) -1s-1 Michaelis-Menten Kinetics

represents the minimal equation needed to describe a simple onesubstrate, one-product reaction catalyzed by an enzyme. This assumes the reverse reaction between E and P is negligible and is a simple, first-order reaction whose rate is determined solely by the concentration of ES and the value of k2. The reaction rate (same as velocity or rate of formation of products) can be written as V = k2[ES] [ES] is usually not a measurable concentration. Easily measurable items are the substrate (or product) and the total concentration of enzyme, which is the sum of the free enzyme and complexed enzyme. That is [E]t = [E] + [ES], where [E]t is total enzyme, [E] is free enzyme, and [ES] is complexed enzyme.

In general, it is incorrect to assume that E and S are in equilibrium with ES, because some ES is continually being drained off to make P. (When k2 is much less than k-1, the assumption is reasonable, however.) In 1925, Briggs and Haldane proposed a model that avoided the equilibrium assumption. It assumes that the more ES that is present, the faster ES will dissociated either to products or back to reactants. Therefore, when the reaction is started by mixing enzymes and substrates, the ES concentration builds up at first, but quickly reaches a steady state, in which it remains almost constant. The steady state will persist until almost all of the substrate has been consumed ( Figure 11.14). If one measures rates only after the steady state has been established and before [ES] has changed much, reaction velocity can be calculated by assuming steady state conditions. In the steady state, rates of formation and breakdown of ES are equal,

Rearranging 11.17 gives equation 11.18,

Combining the three rate constants of equation 11.18 into one, KM, yields KM = (k-1 + k2)/k1 Equation 11.18 becomes KM[ES] = [E][S] Because [E] = [E]t - [ES], KM[ES] = [E]t - [S] - [ES][S] Solving for [ES], [ES] = [E]t[S]/(KM+[S]) Substituting this into the earlier velocity equation, V = k2[E]t[S]/(KM+[S]) This last equation is the Michaelis-Menten equation, and KM is called the Michaelis constant. KM has units of concentration and, because it is a ratio of the rate constants of a reaction, KM is characteristic of the reaction. A given enzyme acting upon a given substrate has a distinct KM.

Figure 11.15 shows a plot of velocity (V) versus substrate concentration ([S]). Note that at high substrate concentrations ([S] >> KM), the velocity approaches a maximum (called Vmax). At this point, the enzyme molecules are saturated with substrate. Note also that the substrate concentration where V = Vmax/2 corresponds to the KM. At Vmax, [S] >> KM, so [S] + KM can be approximated simply by [S]. Thus, the velocity equation simplifies to Vmax = k2[E]t Substituting this back into the velocity equation yields V = Vmax[S]/(KM + [S]) For a multistep reaction more complicated than the one assumed above, the Michaelis-Menten equation must be modified. Consider the reaction

For this reaction, the k2 in the Michaelis-Menten equation must be replaced by a more general constant, called kcat. Here, V = kcat[E]t[S]/( KM + [S]) kcat incorporates the rate constants for all the reactions between ES and E + P. For the two-step reaction above, kcat = k2. For more complex reactions, kcat depends on which steps in the process are rate-limiting. Analysis of Kinetic Data To test whether an enzyme-catalyzed reaction follows the Michaelis-Menten law, an experimenter will usually measure the intitial rates of a series of reactions, all at the same enzyme concentration, but at different substrate concentrations. Because the initial [S] is known precisely, and the change in [S] versus t is almost linear in the initial stages, accurate data for V as a function of [S] can be obtained. A Lineweaver-Burk plot (Figure 11.16) is obtained by inverting the Michaelis-Menten equation to obtain the following:

For a reaction obeying Michaelis-Menten kinetics, plotting 1/V versus 1/[S] should yield a straight line. As seen in Figure 11.16, Vmax and KM can also be obtained easily from such a plot. A disadvantage of the Lineweaver-Burk plot is the long extrapolation necessary to determine KM. One way around this problem is to graph V versus V/[S] (called an Eadie-Hofstee plot). The result,

shown in Figure 11.17, also yields a straight line for reactions obeying Michaelis-Menten kinetics. Here, the slope of the line is equal to -KM. KM, kcat, and kcat /KM KM - The Michaelis constant, KM, is often associated with the affinity of the enzyme for substrate, but this is not always correct. A more accurate statement is that, for reactions obeying MichaelisMenten kinetics, KM is a measure of the substrate concentration required for effective catalysis to occur. That is, an enzyme with a high KM requires a higher substrate concentration to achieve a given reaction velocity than an enzyme with a low KM. Table 11.1 lists values of KM, kcat, and kcat/KM for selected enzymes. kcat - The Michaelis-Menten equation, V = Vmax[S]/(KM + [S]), can be rewritten as V = kcat[E]t[S] / ( KM + [S]) where Vmax = kcat[E]t. kcat incorporates the rate constants for all the reactions between ES and E + P in a multistep enzymatic process. For a two-step reaction, kcat = k2. For more complex reactions, kcat depends on which steps in the process are rate-limiting. kcat gives a direct measure of the catalytic production of product under optimum conditions (saturated enzyme). The units of kcat are seconds-1. The reciprocal of kcat can be thought of as the time required by an enzyme molecule to "turn over" one substrate molecule. Alternatively, kcat measures the number of substrate molecules turned over per enzyme molecule per second. Thus, kcat is sometimes called the turnover number. Some typical turnover numbers are shown in Table 11.1, as well. kcat/KM - This ratio is often thought of as a measure of enzyme efficiency. Either a large value of kcat (rapid turnover) or a small value of KM (high affinity for substrate) makes kcat/KM large. When [S] << KM (dilute solution), equation 11.27 becomes V (kcat/KM)[E][S]

Here, kcat/KM behaves as a second-order rate constant for the reaction between substrate and free enzyme. This ratio is important, for it shows what the enzyme and substrate can accomplish when abundant enzyme sites are available, and it allows direct comparison of the effectiveness of an enzyme toward different substrates. When an enzyme has a choice of two substrates, A or B, present at equal, dilute concentrations,

Table 11.2 shows that when an enzyme has different substrates on which it can work, the range of efficiencies may vary considerably. Note that for chymotrypsin the kcat/KM ratio varies 1million fold. As a second-order rate constant, kcat/KM has a maximum possible value, which is determined by the frequency with which enzyme and substrate molecules can collide. A reaction which attains

such a velocity is said to be "diffusion-limited" because every encounter leads to reaction. If every collision results in formation of an enzyme-substrate complex, diffusion theory predicts that kcat/KM will attain a value of about 108 to 109 (mol/L)-1s-1. The enzymes carbonic anhydrase, fumarase, and triose phosphate isomerase actually approach this limit. Multisubstrate Reactions Most biochemical reactions involve two or more substrates, often resulting in multiple products. An example is proteolysis, which involves two substrates (the polypeptide and water) and two products (the two fragments of the cleaved polypeptide chain). When an enzyme binds two or more substrates and releases multiple products, the order of the steps becomes an important feature of the enzyme mechanism. Several classes of mechanisms include the following: Random substrate binding - In this mechanism, either substrate can be bound first, though in many cases one substrate will be favored for initial binding, and its binding may promote the binding of the other. The general pathway is The phosphorylation of glucose by ATP, catalyzed by hexokinase, appears to follow this mechanism, with some tendency for glucose to bind first. Ordered substrate binding - This mechanism occurs when one substrate must bind before a second one can bind significantly. This mechanism is Ordered substrate binding is often observed in oxidations of substrates by the coenzyme nicotinamide adenine dinucleotide (NAD+). The "ping-pong" mechanism - This occurs when a catalytic sequence of events occurs, such as one substrate is bound, one product is released, a second substrate is bound, and a second product is released. This is shown as where E* is a modified form of the enzyme, often carrying a fragment of the first substrate, S1. A good example is the cleavage of a polypeptide chain by a serine protease, such as trypsin or chymotrypsin. The polypeptide is described here as S = A .B, where A and B designate the C-terminal and N-terminal portions of the chain from the point of cleavage:

Here E*.B and E*.B.H2O indicate covalent intermediates, as in Figure 11.13.

Kinetics of a Complex Reaction - For the cleavage of a substrate by a serine protease, such as chymotrypsin, the step E*.B + H2O -> E*.B.H2O cannot be analyzed. Since the concentration of water is essentially fixed in aqueous solution and is not a variable, the reaction can be written as

Steady state measurements in this case will be insufficient. The steady state velocity is given by

The enzyme obeys Michaelis-Menten kinetics, but kcat, KM, and Ks are defined as kcat = k2k3/(k2 + k3) KM = Ksk3/(k2 + k3) Ks = k-1/k1 Thus, the Michaelis-Menten equation describes the velocity correctly, but the values of kcat and KM depend on the reaction mechanism. To obtain the individual rate constsants in such a case, measurements outside the steady state range must be employed. The kinetics of the hydrolysis of esters by chymotrypsin (the enzyme also works on esters) reveals a rapid concentration increase for a few minutes until about one molecule has been produced per enzyme molecule. Steady state production begins after that point (see Figure 11.18). The initial burst is called presteady state production. For ester hydrolysis, k3 is much smaller than k2. Thus, the acyl intermediate forms quickly on each enzyme molecule, with accompanying release of product A. After this period, however, more A can be formed only after each acyl intermediate breaks down and the enzyme becomes available again. The dissociation of the acyl intermediate is the ratelimiting step. Faster measurement techniques, such as stopped-flow methods, allow measurement of the rate of formation of the ES complex. Measurements of the decay of the acyl intermediates after substrate is exhausted provide k3. Combinations of these methods can be used to obtain all of the constants in equation 11.34. Example rate constants for hydrolysis of two N-acyl amino acid esters by chymotrypsin are given in Table 11.3. Enzyme Inhibition Many different kinds of molecules inhibit enzymes and the inhibition may be reversible or irreversible. Reversible inhibition involves noncovalent binding of the inhibitor and can always be reversed, at least in principle, by removal of the inhibitor. Irreversible inhibition involves a covalent binding of a molecule to the enzyme, which truly incapacitates it.

Reversible Inhibition Competitive inhibition - In this case, the inhibiting compound so closely resembles the substrate for the enzyme that it accepts the molecule to the substrate binding site. However, once bound, the inhibitor cannot be acted on and thus prevents the enzyme from catalyzing the intended reaction. This reaction scheme is depicted as

Here I stands for the inhibitory compound and KI is the dissociation constant for inhibitor binding - KI = [E][I]/[EI]. Now, [E]t = [E] + [ES] + [EI], where [EI] is the concentration of inhibitor-enzyme complex. Then, the velocity is

where

is the apparent KM given by = KM(1 + [I]/KI)

Thus, increasing [I] causes an apparent increase in the KM. The Vmax is unchanged, because as [S] increases relative to a fixed [I], the substrate molecules outcompete the inhibitor for the enzyme's active site. The effect of a competitive inhibitor on a graph of V versus [S] is shown in Figure 11.20a. The system still obeys an equation of the Michaelis-Menten form at a given [I], so the Lineweaver-Burk and Eadie-Hofstee plots are linear, but the KM is altered (Figure 11.20b). By plotting the apparent KM as a function of [I] (Figure 11.20c), one can obtain both KM and KI. A variant of competitive inhibition is nonproductive binding. This occurs when a substrate molecule can fit into the enzyme's binding site in such a way that the normal catalytic event cannot occur. This scheme is as follows:

Here, ES' is the enzyme-substrate complex that cannot lead to product. In this situation, both KM and kcat are modified. Noncompetitive inhibition - This occurs when a molecule or an ion binds to a site on the enzyme other than the active site and modifies kcat (Figure 11.22). Such a compound need not resemble the substrate at all. In fact, it only needs to have a strong affinity for the second binding site. Assuming the inhibitor has equal affinity for E and ES, the scheme is

Mathematical analysis yields equation 11.38. In this case, the KM is unaffected, but the apparent kcat is now given by

=kcat/([1+[I]/KI]). Thus, the apparent kcat decreases with increasing [I]. Vmax is therefore also changed (Figure 11.23a): =kcat(apparent)[E]t = kcat[E]t/(1 + [I]/KI) The effect of noncompetitive binding on a Lineweaver-Burk plot is shown in Figure 11.23b. Both the true kcat and KI may be determined by graphing

versus [I] (Figure 11.23c). The situation is usually more complex than shown here. For example, the complex ESI may also be able to undergo the catalytic process slowly, or the binding of inhibitor may modify both kcat and KM. The latter case is called mixed inhibition. Irreversible Inhibition Irreversible inhibition occurs when substances combine covalently with enzymes so as to inactivate them irreversibly. Almost all irreversible enzyme inhibitors are toxic substances, either natural or synthetic. Some, such as cyanide and penicillin, are shown in Table 11.4. Figure 11.24 depicts the action of the competitive irreversible inhibitor, diisopropyl fluorophosphate (DFP), which reacts with serine groups on a protein to form a covalent adduct. Irreversible inhibitors that strongly resemble the substrate rather than its transition state may be extremely selective. An example is TPCK in Table 11.4, which is an excellent inhibitor for chymotrypsin. When selective irreversible inhibitors are used to label active site residues of an enzyme to aid in their identification, they are called affinity labels. A suicide inhibitor, on the other hand, is an affinity label that is unreactive until it is acted upon by the enzyme, at which point it binds irreversibly.