LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOLUME 1. THE FOUNDATIONS OF BIOCHEMISTRY: STRUCTURE AND CATALYSIS - 2011

PART I. STRUCTURE AND CATALYSIS

6. ENZYMES

6.4. Examples of Enzymatic Reactions

Up to this point, we have discussed the General Principles of catalysis and introduced several kinetic parameters to describe enzyme action. Now we turn to specific Examples of enzymatic reaction mechanisms.

Fully elucidating the MECHANISM OF ACTION of an isolated enzyme requires identifying all its substrates, Cofactors, products, and regulatory molecules. Furthermore, we must determine: 1) the order of steps in the enzymatic reaction, 2) The Structure of each reaction intermediate, 3) the rates of interconversion between intermediates, 4) the structural relationships between the enzyme and each intermediate, and 5) the energy contribution of every bond formed in intermediate complexes and transition states. To date, there is probably not a single enzyme for which Answers to all these questions are known. However, decades of research have accumulated a vast body of information—in some cases quite detailed—concerning the catalytic mechanisms of hundreds of Enzymes.

In the sections that follow, we examine the mechanisms of four enzymes: Chymotrypsin, hexokinase, enolase, and Lysozyme. These examples certainly cannot capture the full diversity of enzymatic reactions. They are chosen in part because they are among the most thoroughly studied enzymes, and additionally because they serve to illustrate several general principles described in this chapter. Throughout our Discussion of enzyme mechanisms, we will also highlight the key experiments used to establish them. Using chymotrypsin as an example, we will revisit the standard rules applied to describe enzymatic mechanisms. Many mechanistic details and experimental findings must, of course, be omitted, as no single book could contain the rich history of experiments dedicated to these enzymes. Furthermore, we will not specifically address The Role of Coenzymes in enzymatic reactions here; various coenzyme Functions will be covered in greater detail in Part II.

The Mechanism of Chymotrypsin action involves acylation and deacylation of a Serine residue

Bovine pancreatic chymotrypsin (Mr = 25,191) is a protease—an enzyme that catalyzes the Hydrolysis of peptide bonds. Specifically, this protease cleaves peptide bonds on the carboxyl side of aromatic amino acid residues (Trp, Phe, and Tyr). Figure 6-18 illustrates the three-dimensional structure of chymotrypsin with the functional groups in its Active Site highlighted. The reaction catalyzed by this enzyme exemplifies transition-state stabilization and serves as a classic model of general acid-base and Covalent Catalysis.

Class="center">Figure 6-18. Structure of chymotrypsin (PDB ID 7GCH). (a) Introduction/19.html">Primary Structure of the protein, indicating Disulfide Bonds and catalytically crucial amino acid residues. The protein consists of three polypeptide chains held together by disulfide bridges (the numbering of chymotrypsin residues, with "missing" residues 14, 15, 147, and 148, is explained in Figure 6-38). In the three-dimensional structure, The amino acid residues that form the active site are clustered together. (b) Surface representation of the protein. The pocket that binds the aromatic side chain of the substrate amino acid residue is highlighted in green. Key active site residues—Ser¹⁹⁵, His⁵⁷, and Asp¹⁰²—are shown in red. The catalytic roles of these residues are depicted in Figure 6-21. (c) Ribbon diagram of the protein backbone. Disulfide bonds are colored yellow, and the three polypeptide chains are shown in the same colors as in panel (a). (d) Active-site closure upon substrate binding (the bulk of the protein is shown in green). Two active-site residues, Ser¹⁹⁵ and His⁵⁷, are partially visible (red). The hydroxyl group of Ser¹⁹⁵ attacks the carbonyl group of the substrate (carbonyl oxygen is colored purple); the resulting negative charge on the oxygen is stabilized in a pocket formed by amide backbone nitrogens (including that of Ser¹⁹⁵, shown in orange), as explained in Figure 6-21. The aromatic side chain and the amide nitrogen of the scissile peptide bond are colored blue (projecting toward the reader and obscuring the rest of the substrate polypeptide chain).

Chymotrypsin accelerates the hydrolysis of peptide bonds by a factor of at least 10⁹. Rather than directly attacking the peptide bond with a Water molecule, the enzyme participates in The formation of a transient acyl-enzyme intermediate. This reaction proceeds in two distinct phases. In the acylation phase, the peptide bond is cleaved, and an ester bond is formed between the peptide carbonyl group and the enzyme. In the deacylation phase, this ester bond is hydrolyzed, releasing the free enzyme.

The existence of a covalent acyl-enzyme intermediate was demonstrated using pre-steady-state kinetics. Chymotrypsin hydrolyzes not only Peptides but also small ester and amide molecules. These reactions proceed much more slowly than peptide hydrolysis because the binding energy for small substrates is substantially lower, making such reactions easier to study. In 1954, B. S. Hartley and B. A. Kilby discovered that The rate of p-nitrophenyl acetate hydrolysis catalyzed by chymotrypsin, monitored by the formation of p-nitrophenol, was initially very high before dropping to a slower steady-state rate (Figure 6-19). By extrapolating back to time zero, they concluded that the burst phase corresponds to the formation of one molecule of p-nitrophenol per enzyme molecule. Hartley and Kilby hypothesized that this reaction involves rapid acylation of all available enzyme (with the release of p-nitrophenol) followed by its slow deacylation. Similar results have since been obtained for many Other Enzymes. The ability to detect a rapid initial phase of a reaction is yet another example of how kinetic Methods can be used to identify reaction intermediates.

Figure 6-19. Use of pre-steady-state kinetics to demonstrate the formation of an acyl-enzyme intermediate. The hydrolysis of p-nitrophenyl acetate was monitored by the appearance of the colored product p-nitrophenol. Initially, the rate of p-nitrophenol production is nearly stoichiometric with the Enzyme Concentration, reflecting the rapid acylation phase. The rate then drops as the release of the enzyme becomes limited by the slower deacylation step.

Further insights into The Mechanism of chymotrypsin action came from analyzing The Effect of pH on reaction velocity. The dependence of the rate of chymotrypsin-catalyzed substrate hydrolysis on pH typically exhibits a bell-shaped curve (Figure 6-20). The reaction rates in Figure 6-20a were determined at a low (nonsaturating) Substrate Concentration and therefore reflect the ratio r_cat/K_m. This curve can be dissected into its constituent parts by determining the maximum velocity at each pH and plotting r_cat versus pH (Figure 6-20b). Once K_m is determined at each pH, a plot of 1/K_m versus pH can also be constructed (Figure 6-20c). Kinetic and structural analyses revealed that the variation in r_cat is linked to the ionization state of His⁵⁷. The decrease in r_cat at lower pH values results from the protonation of His⁵⁷, which can no longer Abstract a proton from Ser¹⁹⁵ in the initial step of the reaction (Figure 6-21). This drop in rate highlights the essential role of general Acid-Base Catalysis in the mechanism of chymotrypsin. Changes in 1/K_m reflect The ionization of the α-amino group of Ile¹⁶, located at the N-terminus of one of chymotrypsin's three polypeptide chains. This group forms a salt bridge with Asp¹⁹⁴, which stabilizes the active conformation of the enzyme. When this group loses a proton at higher pH, the salt bridge disrupts, and the resulting conformational changes close off the hydrophobic pocket that accommodates the aromatic side chain of the substrate (Figure 6-18). As a result, the substrate can no longer bind effectively, which is manifested as an increase in the Michaelis constant.

Figure 6-20. pH dependence of chymotrypsin activity. (a) The rate of chymotrypsin-catalyzed substrate hydrolysis as a function of pH displays a bell-shaped curve with a maximum at pH 8.0. The velocity v was measured at a low substrate concentration and thus reflects the ratio r_cat/K_m. Kinetic methods allow r_cat and K_m to be determined separately and plotted individually as a function of pH (b and c). These plots show that the transition at pH > 7 is linked to A change in r_cat, whereas the transition at pH > 8.5 is linked to a change in K_m. Kinetic and structural studies indicate that these transitions reflect Changes in the ionization state of the His⁵⁷ side chain (in the absence of substrate binding) and the α-amino group of Ile¹⁶ (at the N-terminus of chain B), respectively. For optimal enzymatic activity, His⁵⁷ must be unprotonated, and Ile¹⁶ must be protonated.

The nucleophile in the acylation step is the oxygen atom of the Ser¹⁹⁵ residue. (Proteases that rely on a catalytic serine residue are known as serine proteases.) The pK_a of a standard serine hydroxyl group is normally too high for the unprotonated nucleophilic form to exist in significant concentrations at physiological pH. However, within the chymotrypsin molecule, Ser¹⁹⁵ is hydrogen-bonded to His⁵⁷ and Asp¹⁰² in what is known as a catalytic triad. When a peptide substrate binds to chymotrypsin, a subtle conformational change shortens the Hydrogen bond between His⁵⁷ and Asp¹⁰², resulting in a stronger interaction (a low-barrier hydrogen bond). This, in turn, raises the pK_a of His⁵⁷ from ~7 (in free Histidine) to >12, enabling the histidine to act as a potent general base that extracts a proton from the Ser¹⁹⁵ hydroxyl group. Deprotonation prevents the formation of an energetically unfavorable positive charge on the Ser¹⁹⁵ oxygen and converts the serine side chain into a powerful nucleophile. Later in the reaction pathway, His⁵⁷ acts as a proton donor, protonating the amino group of the leaving group.

When the oxygen of the Ser¹⁹⁵ residue attacks the carbonyl carbon of the substrate, a short-lived tetrahedral intermediate is formed in which the carbonyl oxygen bears a negative charge (Figure 6-21). This charge, localized within an enzyme pocket known as the oxyanion hole, is stabilized by Hydrogen Bonds donated by backbone amide groups of the chymotrypsin polypeptide. One of these hydrogen bonds (involving Gly¹⁹³) forms exclusively in this intermediate and in the transition states leading to and from it; this interaction lowers the activation energy required to reach these states. This provides a clear illustration of the role of binding energy in catalysis.

THE PRINCIPLE OF enzyme transition-state complementarity in Enzymatic Catalysis is further discussed in Box 6-3.

Unraveling Reaction Mechanisms

Figure 6-21. Mechanism of chymotrypsin-catalyzed peptide bond hydrolysis. The reaction occurs in two stages. The First stage (steps 1–3) involves the formation of a covalent acyl-enzyme intermediate coupled with peptide bond Cleavage. The deacylation stage (steps 4–7) regenerates the free enzyme. This latter stage is essentially the reverse of acylation, with a water molecule playing the role of the amino component of the substrate.

* The tetrahedral intermediates formed in chymotrypsin-catalyzed reactions are sometimes referred to as transition states, which can cause confusion. An intermediate is a chemical species with a finite lifetime exceeding that of a single molecular vibration (≈10^-13 s). A Transition State is a point along the reaction coordinate with maximum Free energy and is not characterized by a lifetime. The tetrahedral intermediates formed in the two steps of this reaction are very similar in both energy state and structure, with transition states arising during the formation and breakdown of these intermediates. Thus, an intermediate represents an obligatory stage of bond formation, whereas a transition state is a snapshot of the reaction process itself. In the case of chymotrypsin, the close relationship between the intermediate and the transition states sometimes leads to the blurring of these concepts. Furthermore, the interaction of the negatively charged oxygen with the amide nitrogen atoms in the oxyanion hole—often described as transition-state stabilization—also plays a role in stabilizing the intermediate in this instance. Not all intermediates are short-lived enough to resemble transition states. The acyl-enzyme intermediate of chymotrypsin is a much more stable species that can be readily detected and studied, and it is never confused with a transition state.

Evidence for Enzyme Complementarity to the Transition State

The transition state of a reaction is notoriously difficult to study directly because of its extremely fleeting nature. Nevertheless, understanding the mechanism of catalysis requires analyzing the interactions between the enzyme and this transient substrate state during the course of the reaction. Enzyme-transition state complementarity can be considered a prerequisite for catalysis, as a lowering of the transition-state energy barrier occurs only when catalysis takes place. How can one obtain evidence for complementarity between an enzyme and a transition state? Fortunately, researchers have at their disposal a variety of classic and modern approaches to address this challenge, each contributing to the foundation of our understanding of enzyme action.

Structure-Activity Relationships

If an enzyme is complementary to the reaction's transition state, certain functional groups on both the enzyme and the substrate must interact preferentially in the transition state rather than in the ES complex. Alterations to these groups should have little effect on complex formation and, consequently, should not significantly alter constants such as the dissociation constant (K_d, or K_m when K_d = K_m), which characterize the E + S ⇄ ES equilibrium. However, modifying those same groups should profoundly impact the overall reaction rate (i.e., r_cat or the r_cat / K_m ratio), because substrate binding fails to engage the interactions necessary to lower the activation energy.

Kinetic experiments using a series of related chymotrypsin substrates (Fig. 1) provide an exceptional illustration of this effect. Normally, chymotrypsin hydrolyzes a peptide bond adjacent to an aromatic amino acid. Figure 1 shows the structures of low-molecular-weight substrates used as models for natural substrates like long peptides or Proteins. The chemical group added sequentially in each subsequent substrate (from A through B to C) is highlighted in color. As the table demonstrates, adding groups to the substrate molecule had almost no effect on the Michaelis constants, yet it dramatically increased r_cat and r_cat / K_m. This is precisely the outcome expected if enzyme-substrate interactions contribute more heavily to transition-state stabilization. These findings also reveal that reaction rates depend heavily on enzyme-substrate interactions occurring at sites remote from the reacting groups. The mechanism of chymotrypsin action is discussed in greater detail in the main text.

Fig. 1. Effect of minor variations in substrate structure on the kinetic parameters of chymotrypsin-catalyzed amide hydrolysis.

An additional analytical method involves modifying the enzyme itself to abolish specific enzyme-substrate interactions. This can be achieved by introducing targeted amino acid replacements via Site-Directed Mutagenesis (Figs. 9-11). The results of such experiments further underscore the critical role of binding energy in stabilizing the transition state.

Transition-State Analogs

Even when a transition state cannot be observed directly, a chemist can often deduce its approximate structure based on reaction mechanism data. By definition, a transition state is temporary and so unstable that direct measurement of interactions between the intermediate and the enzyme is impossible. In some cases, however, stable molecules can be synthesized whose structures mimic the transition state; these are known as transition-state analogs. In theory, such compounds should bind to the enzyme much more tightly than the substrate does in the ES complex, because they fit the active site better (i.e., they form a greater number of weak interactions) than the substrate itself. METABOLISM/2.html">THE CONCEPT OF designing transition-state analogs was proposed by Pauling in the 1940s and has since been realized for several enzymes. A limitation of this approach is that analogs cannot perfectly mimic the transition state in every detail. Nevertheless, some bind to the enzyme 10² to 10⁶ times more tightly than normal substrates, providing compelling evidence that the enzyme's active site is indeed complementary to the transition state. This very principle is widely applied in modern pharmaceutical development to design novel drugs. Potent anti-HIV therapeutics—protease inhibitors—were engineered as tightly binding transition-state analogs designed to fit the Active Site of the HIV protease.

Catalytic Antibodies

If a transition-state analog can be synthesized for a reaction S ⇄ P, then antibodies that bind tightly to these analogs might prove capable of catalyzing the reaction S -> P. Antibodies (or IMMUNOGLOBULINS, Figs. 5-23) play a central role in The Immune System. When a transition-state analog is used as an epitope to stimulate antibody production, the resulting antibodies are potential catalysts for the corresponding reaction. The Use of such "catalytic antibodies" (often called abzymes, a portmanteau of antibody and enzyme) was first proposed by W. P. Jencks in 1969 and became practical with the advent of laboratory techniques capable of yielding sufficient quantities of identical antibodies directed against a single specific antigen (Monoclonal Antibodies, Ch. 5).

Pioneering work in the laboratories of Richard Lerner and Peter Schultz led to the isolation of several monoclonal antibodies that catalyze the hydrolysis of esters or carbonates (Fig. 2). In these reactions, the nucleophilic attack of water (OH^-) on the carbonyl carbon generates a tetrahedral transition state in which a partial negative charge is concentrated on the carbonyl oxygen. Phosphonate esters structurally and electronically resemble the transition state of ester hydrolysis, making them well-suited for use as transition-state analogs. Similarly, phosphonate and phosphate esters are employed for carbonate hydrolysis. Antibodies that bind tightly to these Phosphonates or phosphates were found to accelerate the corresponding ester or carbonate hydrolysis reactions by a factor of 10³ to 10⁴. Structural analysis of several such catalytic antibodies revealed that the side chains of specific amino acid residues are positioned in a way that allows them to interact favorably with the substrate in its transition state.

Fig. 2. Proposed transition states for ester and carbonate hydrolysis reactions. Phosphonate and phosphate esters serve as effective transition-state analogs for these reactions, respectively.

Although catalytic antibodies typically do not match the catalytic efficiency of natural enzymes, their potential medical and industrial Applications are evident. For instance, it has been proposed to utilize catalytic antibodies engineered to degrade cocaine as a therapeutic strategy for cocaine addiction.

Induced fit in Substrate Binding to Hexokinase

Yeast hexokinase (M_r = 107,862) catalyzes a reversible two-substrate reaction:

ATP and ADP invariably bind to enzymes as complexes with Mg²⁺ ions.

The C-6 hydroxyl group of glucose (to which the γ-phosphoryl group of ATP is transferred during the enzymatic reaction) has a reactivity similar to that of water, and water readily gains access to the enzyme's active site. Yet, hexokinase enhances the rate of the glucose reaction by a factor of 10⁶. The enzyme's ability to discriminate between water and glucose stems from conformational changes within its structure triggered by the binding of the "correct" substrate (Fig. 6-22). Thus, the mechanism of hexokinase action is a textbook example of induced fit. In the absence of glucose, the enzyme exists in an inactive conformation with its active-site amino acid residues held in non-reactive positions. Upon the binding of glucose (but not water) and Mg•ATP, the resulting binding energy drives a conformational shift that switches hexokinase into its active state.

Fig. 6-22. Induced fit in hexokinase. (a) Hexokinase adopts a U-shaped conformation (PDB ID 2YHX). (b) The binding of D-glucose (highlighted in red) induces a conformational change characterized by the closing of the two lobes of the molecule (PDB ID 1HKG and PDB ID 1GLK).

This model has been strongly supported by kinetic studies. Xylose, a five-carbon sugar stereochemically similar to glucose but lacking one carbon atom, binds to hexokinase yet cannot be phosphorylated. Nevertheless, adding xylose to the reaction mixture accelerates the rate of ATP hydrolysis. Apparently, xylose binding is sufficient to drive hexokinase into its active conformation, causing the "fooled" enzyme to phosphorylate water. This hexokinase reaction also demonstrates that Enzyme Specificity does not always rely on binding a single unique substance. In this case, specificity resides not in the Formation of the ES complex, but rather in the relative rates of the subsequent catalytic steps. While water faces no barrier to entering the active site, the reaction rate increases dramatically only in the presence of a functional phosphoryl group acceptor (glucose).

Induced fit is only one aspect of the hexokinase catalytic mechanism. Like chymotrypsin, this enzyme employs multiple catalytic strategies. For example, active-site amino acid residues—those that assume the correct orientation as a result of conformational changes upon substrate binding—participate in general acid-base catalysis and transition-state stabilization.

The Enolase Reaction Mechanism Requires Metal Ions

Another glycolytic enzyme, enolase, catalyzes the reversible dehydration of 2-phosphoglycerate to form phosphoenolpyruvate:

Yeast enolase (M_r = 93,316) is a dimer, with each subunit consisting of 436 amino acid residues. The enzymatic reaction involving enolase illustrates one type of metal ion catalysis and provides yet another example of general acid-base catalysis and transition-state stabilization. The reaction proceeds in two steps (Fig. 6-23a). First, the Lys³⁴⁵ residue acts as a general base, abstracting a proton from the C-2 atom of 2-phosphoglycerate. Next, the Glu²¹¹ residue acts as a general acid, donating a proton to the departing OH group. The proton at the C-2 position of 2-phosphoglycerate is not very acidic and is therefore not easily abstracted. However, within the active site of the enzyme, 2-phosphoglycerate participates in rather strong ionic interactions with two bound Mg²⁺ ions (Fig. 6-23b). As a result, the proton at the C-2 atom becomes more acidic (its pK_a decreases) and is removed more readily. Hydrogen bonds involving other active-site amino acid residues also contribute to the catalytic mechanism. These various interactions lead to the effective stabilization of both the enolic intermediate and the transition state leading to its formation.

Fig. 6-23. Reaction mechanism. The two-step reaction catalyzed by enolase. (a) Mechanism of The conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate. The carboxyl group of 2-PG is coordinated by two magnesium ions in the enzyme's active site. (b) Positions of the substrate, Mg²⁺ ions, Lys³⁴⁵, and Glu²¹¹ in the active site of enolase. Hydrogen atoms are not shown. All oxygen atoms in the 2-PG molecule are depicted in pink, and the phosphorus atom is in orange (PDB ID 1ONE).

The Mechanism of Lysozyme Action Involves Two Successive Nucleophilic Substitution Steps

Lysozyme is a natural antibacterial agent found in tears and egg white. Hen egg-white lysozyme (M_r = 14,296) is a monomer consisting of 129 amino acid residues. In 1965, it became the first enzyme whose three-dimensional structure was determined by David Phillips and his coworkers. The lysozyme molecule is held together by four disulfide bonds and features a long cleft where its active site is located (Fig. 6-24a). Over five decades of active research on lysozyme have yielded a detailed picture reflecting its structure and activity, serving as a landmark in The Development of biochemical science.

Fig. 6-24. Structure of hen egg-white lysozyme and the reaction it catalyzes. (a) Ribbon model of the Cell/13.html">Protein Structure: the Glu³⁵ and Asp⁵² residues in the active site are shown as blue rods, and the bound substrate is highlighted in red (PDB ID 1LZE). (b) Reaction catalyzed by hen egg-white lysozyme. A fragment of peptidoglycan is shown, indicating the binding sites within the enzyme molecule, designated by letters from A to F. The red arrow points to the cleavable C–O glycosidic bond located between the sugar residues bound in sites D and E. The inset shows the reaction itself; oxygen originating from a water molecule is highlighted in red. Abbreviations used: Mur2Ac, N-acetylmuramic acid; GlcNAc, N-acetylglucosamine; RO, lactic acid residue; NAc and AcN, N-acetyl group.

The substrates for lysozyme are peptidoglycans, CARBOHYDRATES found in The Cell walls of many Bacteria (see Fig. 20-31). Lysozyme cleaves the β1 -> 4 glycosidic C–O bond (see p. 348) between Two Types of sugar residues in the molecule: N-acetylmuramic acid (Mur2Ac) and N-acetylglucosamine (GlcNAc), often referred to as NAM and NAG, respectively (Fig. 6-24b). Six alternating residues of these sugars in the peptidoglycan molecule bind

in the enzyme's active site at positions designated by letters from A to F. Model-building studies have shown that the side chain of N-acetylmuramic acid prevents this residue from binding in positions C and E, restricting it to positions B, D, or F. Upon peptidoglycan binding, only a single glycosidic bond is cleaved—the one connecting the N-acetylmuramic acid residue at position D and the N-acetylglucosamine residue at position E. The key amino acid residues of the enzyme are Glu³⁵ and Asp⁵² (Fig. 6-25a). The reaction proceeds via a nucleophilic substitution mechanism: the OH group of water replaces the N-acetylglucosamine residue at the C-1 atom of N-acetylmuramic acid.

Fig. 6-25. Reaction mechanism. Action of lysozyme. In this reaction, the water molecule adding to the C-1 atom of Mur2Ac in the product has the same configuration as the original glycosidic bond. Thus, the reaction proceeds with retention of configuration. (a) Two mechanisms proposed to describe this reaction. The S_N1-type mechanism proposed by Phillips is shown on the left. The S_N2-type mechanism shown on the right is in better agreement with modern experimental data. (b) Surface of the lysozyme active site with a ball-and-stick model of the covalent enzyme-substrate intermediate. Active-site side chains are shown as ball-and-stick structures emerging from the ribbon backbone (PDB ID 1H6M).

It seemed that following the identification of active-site amino acid residues and the detailed elucidation of the protein structure in the 1960s, the mechanism of lysozyme action had become clear. However, a final understanding of this mechanism was reached only nearly forty years later. There are two possible reaction pathways leading to the formation of the glycosidic bond hydrolysis products. Phillips and coworkers proposed that the reaction proceeds via a dissociative S_N1-type pathway (Fig. 6-25a, left), in which GlcNAc first dissociates (step 1) to form a carbocation intermediate. In this scheme, the departing GlcNAc is protonated via a general acid catalysis mechanism involving Glu³⁵, which is located in a hydrophobic pocket that confers an unusually high pK_a on its carboxyl group. The carbocation is stabilized by a Resonance structure involving the oxygen of the six-membered ring, as well as Electrostatic Interactions with the negative charge on the neighboring Asp⁵². In step 2, water attacks the C-1 atom of Mur2Ac, yielding the product. An alternative mechanism (Fig. 6-25a, right) involves two successive S_N2-type substitution steps. In step 1, Asp⁵² attacks the C-1 atom of Mur2Ac and displaces GlcNAc. As in the first mechanism, Glu³⁵ acts as a general acid and protonates the departing GlcNAc. In step 2, water attacks the C-1 atom of Mur2Ac, resulting in the displacement of Asp⁵² and the formation of the product.

The mechanism proposed by Phillips (S_N1-type) was accepted by most researchers for over three decades. Nevertheless, some controversies persisted, and work in this area continued. Analytical Methods sometimes evolve too slowly, making it difficult to design an experiment that provides a definitive answer to a given question. Some arguments against the Phillips hypothesis cast doubt upon it, but they were not particularly compelling. For example, the half-life of the glycosyl cation was estimated to be 10^-12 s—longer than a molecular vibration period, yet insufficient for the diffusion of other molecules. More importantly, lysozyme belongs to a family of enzymes that retain the anomeric configuration of the product identical to that of the substrate (for carbohydrate anomeric configurations, see Chapter 7). All these enzymes are known to catalyze reactions that proceed via a covalent intermediate, analogous to the one that would arise in the alternative (S_N2-type) mechanism of lysozyme action. Thus, the Phillips mechanism conflicted with experimental data for closely related proteins.

An experiment conducted by Stephen Withers and his coworkers in 2001 finally tipped the scales in favor of the S_N2 mechanism. Using a mutant enzyme (in which Glu³⁵ was replaced by Gln) and an artificial substrate to slow down the key reaction steps, they successfully stabilized the covalent intermediate and studied it directly using mass spectrometry and X-ray crystallography (Fig. 6-25b).

Can the mechanism of lysozyme action be considered definitively proven? No. As Albert Einstein noted, one of the CHARACTERISTICS OF THE scientific approach to problem-solving is that "no amount of experimentation can ever prove me right; a single experiment may at any time prove me wrong." In the case of the lysozyme mechanism, one might object (and objections have been raised) that the use of artificial substrates with fluorine atoms at the C-1 and C-2 positions, introduced to stabilize the covalent intermediate, could have altered the reaction pathway. The strongly electronegative fluorine could have destabilized the already electron-deficient carbenium ion intermediate generated via the S_N1 mechanism. Even so, the S_N2 mechanism currently provides the best account of the available experimental data.

Understanding Enzyme Mechanisms Stimulates Medical Advances

■ Most drugs used to treat various diseases—ranging from headaches to HIV infection—are Enzyme Inhibitors. Here we consider two examples: the antibiotic penicillin (and its derivatives) and protease inhibitors used in HIV Treatment; these agents act as irreversible enzyme inhibitors.

Penicillin was discovered by Alexander Fleming in 1928, but another 15 years passed before this relatively unstable substance was studied well enough to be used as a medicine for bacterial infections. Penicillin blocks the synthesis of peptidoglycan (see Chapter 20; Fig. 20-32), a major component of the rigid Cell wall that protects bacteria from osmotic lysis. Peptidoglycan is formed by the cross-linking of Polysaccharides and peptides via the enzyme transpeptidase (Fig. 6-26). It is this reaction that is inhibited by penicillin and its analogs (Fig. 6-27a), which mimic the D-Ala-D-Ala segment in the peptidoglycan precursor sequence. The peptide bond in the precursor is replaced by a reactive β-lactam ring. When penicillin binds to transpeptidase, the active-site serine residue attacks the carbonyl group of the β-lactam ring, forming a covalent penicillin-enzyme adduct. However, the leaving group remains attached to the β-lactam ring moiety (Fig. 6-27b). This covalent complex irreversibly inactivates the enzyme. This, in turn, blocks Bacterial cell wall synthesis, and most bacteria die as their fragile inner membrane ruptures under osmotic Shock.

Fig. 6-26. The transpeptidase-catalyzed reaction. The cross-linking of two precursors into a larger peptidoglycan polymer molecule involves an active-site serine residue of the enzyme, operating via a covalent catalysis mechanism reminiscent of the reaction catalyzed by chymotrypsin. Interestingly, peptidoglycan is one of the few natural compounds in which D-amino acid residues are found. The active-site serine attacks the carbonyl group of the peptide bond between two D-Ala residues, forming an ester linkage between the substrate and the enzyme while releasing the terminal D-Ala residue. Next, the amino group of the second precursor attacks the ester linkage, displacing the enzyme and thereby cross-linking the two precursors.

Fig. 6-27. Inhibition of transpeptidase by β-lactam Antibiotics. (a) β-Lactam antibiotics consist of a five-membered thiazolidine ring fused to a four-membered β-lactam ring. The latter is a strained structure containing an amide bond that plays a critical role in halting peptidoglycan synthesis. The R group varies among different Penicillins. Penicillin G was the first to be isolated and remains one of the most effective, yet it is destroyed by Stomach acid and therefore must be administered via injection. Penicillin V is nearly as active and is acid-stable, making it suitable for oral administration. Amoxicillin is effective against a broad spectrum of infections, can be taken orally, and is consequently the most frequently prescribed β-lactam antibiotic. (b) Nucleophilic attack by the active-site Ser residue of transpeptidase on the amide group of the β-lactam ring leads to the formation of an acylated enzyme. The hydrolysis of this compound is extremely slow, rendering the adduct formation virtually irreversible and leaving the transpeptidase inactivated.

The Human Use of penicillin and its derivatives has driven the evolution of pathogenic bacterial strains capable of synthesizing β-lactamases (Fig. 6-28a)—enzymes that cleave and thereby inactivate β-lactam antibiotics. As a result, these bacteria have acquired resistance to this class of antibiotics. The genes encoding these enzymes spread rapidly through bacterial populations under the selective pressure imposed by the widespread (and sometimes excessive) use of β-lactam antibiotics. In response to The Emergence of β-lactamases, pharmaceutical chemists developed compounds such as clavulanic acid, a mechanism-based (suicide) inhibitor that irreversibly inactivates β-lactamases (Fig. 6-28b). The structure of clavulanic acid closely mimics that of a β-lactam antibiotic, enabling it to form a covalent adduct with the active-site serine residue of the β-lactamase. This triggers specific conformational rearrangements that generate a highly reactive intermediate, which is subsequently attacked by another active-site nucleophile, resulting in the permanent acylation and inactivation of the enzyme. Augmentin™, a widely prescribed medication, is a combination of amoxicillin and clavulanic acid. The "chemical warfare" between humans and bacteria is ongoing; strains of pathogenic bacteria resistant to both amoxicillin and clavulanic acid have already been discovered (indicating Mutations in the β-lactamase molecule that prevent its interaction with clavulanic acid). Undoubtedly, the development of novel antibiotics will accelerate even further in the near future.

Fig. 6-28. β-Lactamases and their inhibitors. (a) β-Lactamases catalyze the Cleavage of the β-lactam ring in β-lactam antibiotics, thereby inactivating them. (b) Clavulanic acid is a suicide inhibitor whose action relies on the normal chemical mechanism of generating reactive species within the active site, which then react with other active-site residues, leading to irreversible enzyme acylation.

Antiviral drugs provide another striking example of modern pharmacotherapy. HUMAN IMMUNODEFICIENCY VIRUS (HIV) is the CAUSATIVE AGENT OF Acquired Immunodeficiency Syndrome (AIDS). Estimates indicate that in 2005, between 37 and 45 million people were living with the virus, with 3.9 to 6.6 million new infections and over 2.4 million deaths occurring annually. AIDS was first recognized as an epidemic in the 1980s.

Shortly thereafter, the human immunodeficiency virus was identified as a member of the retrovirus family. Retroviral genomes consist of RNA, and the enzyme Reverse Transcriptase uses this RNA template to synthesize a complementary DNA sequence. Understanding the mechanisms of HIV action and developing therapeutic strategies against the infection are grounded in years of foundational research conducted on other Retroviruses. A retrovirus like HIV possesses a relatively simple life cycle (see Fig. 26-33). Its genomic RNA is converted into a DNA double helix by reverse transcriptase (see Chapter 26). This double-stranded DNA is subsequently integrated into the host cell's nuclear chromosome through the action of the enzyme integrase (see Chapter 25). The integrated viral genome can remain latent indefinitely, or it can be transcribed back into RNA, which is then translated into the proteins required for the assembly of new Viral Particles. Most viral genes are translated into large polyproteins that are cleaved by HIV protease into the individual functional proteins necessary for viral assembly (see Fig. 26-34). Only three Key Enzymes are involved in this cycle: reverse transcriptase, integrase, and protease, each of which represents a potential drug target.

There are four major subclasses of proteases. Serine proteases, such as Chymotrypsin and Trypsin, and Cysteine proteases (in which Cys plays a catalytic role analogous to that of Ser) form covalent enzyme-substrate intermediates, whereas aspartyl proteases and metalloproteases do not. HIV protease is an aspartyl protease. Two active-site Asp residues facilitate the direct nucleophilic attack of a water molecule on the peptide bond to be cleaved (Fig. 6-29). The initial attack of the water molecule on the carbonyl carbon of the peptide bond generates an unstable tetrahedral intermediate, analogous to that observed in the chymotrypsin reaction. Structurally and energetically, this intermediate closely resembles the transition state of the reaction. HIV protease inhibitor drugs form noncovalent complexes with the enzyme, but bind so tightly that they function essentially as irreversible inhibitors. This high binding affinity is due in part to the fact that these compounds act as transition-state analogs (see Box 6-3). We highlight this fact to reiterate that the catalytic principles explored in this chapter are far from abstract concepts—their Practical Application saves lives.

Fig. 6-29. Catalytic Mechanism of HIV protease. Two active-site Asp residues (from different subunits) function as general acid-base catalysts, facilitating the attack of a water molecule on the peptide bond. The unstable tetrahedral reaction intermediate is highlighted in pink.

HIV protease preferentially cleaves peptide bonds between Phe and Pro residues. The active site of the enzyme features a hydrophobic pocket that accommodates the aromatic side chain adjacent to the scissile bond. The structures of several HIV protease inhibitors are illustrated in Fig. 6-30. Although these structures appear diverse, they share a common feature: a backbone hydroxyl group positioned adjacent to a benzene-containing side chain. This architecture allows the benzene ring to fit snugly into the hydrophobic binding pocket. The neighboring hydroxyl group mimics the negatively charged oxygen of the tetrahedral intermediate formed during the normal catalytic reaction, thereby functioning as a transition-state analog. The remainder of each inhibitor molecule is meticulously designed to maximize shape complementarity with the surface contours of the enzyme, further enhancing binding affinity.

Fig. 6-30. HIV protease inhibitors. The hydroxyl group (highlighted in red) mimics the carbonyl oxygen of the tetrahedral intermediate, creating a transition-state analog. The adjacent benzene ring (blue) assists in properly positioning the drug within the enzyme's active site.

The development of such drugs has dramatically increased both the lifespan and the quality of life for millions of individuals affected by AIDS. ■

Summary of Section 6.4 Examples of Enzymatic Reactions

■ Chymotrypsin is a serine protease whose catalytic mechanism is well-characterized. It incorporates elements of general acid-base catalysis, covalent catalysis, and transition-state stabilization.

■ The action of hexokinase serves as an excellent illustration of how substrate-binding energy is harnessed via induced fit.

■ The reaction catalyzed by enolase provides a classic example of metal ion catalysis.

■ Lysozyme catalysis relies on covalent and general acid-base mechanisms, proceeding through two sequential nucleophilic substitution steps.

■ Understanding enzyme mechanisms enables the rational design of targeted pharmaceutical inhibitors.