9.1.1 Chymotrypsin Possesses a Highly Reactive Serine Residue

A number of proteolytic enzymes participate in the breakdown of proteins in the digestive systems of mammals and other organisms. One such enzyme, chymotrypsin, cleaves peptide bonds selectively on the carboxylterminal side of the large hydrophobic amino acids such as tryptophan, tyrosine, phenylalanine, and methionine (Figure 9.1). Chymotrypsin is a good example of the use of covalent modification as a catalytic strategy. The enzyme employs a powerful nucleophile to attack the unreactive carbonyl group of the substrate. This nucleophile becomes covalently attached to the substrate briefly in the course of catalysis.

Figure 9.1

Specificity of Chymotrypsin. Chymotrypsin cleaves proteins on the carboxyl side of aromatic or large hydrophobic amino acids (shaded yellow). The likely bonds cleaved by chymotrypsin are indicated in red.

What is the nucleophile that chymotrypsin employs to attack the substrate carbonyl group? A clue came from the fact that chymotrypsin contains an extraordinarily reactive serine residue. Treatment with organofluorophosphates such as diisopropylphosphofluoridate (DIPF) (Section 8.5.2) was found to inactivate the enzyme irreversibly (Figure 9.2). Despite the fact that the enzyme possesses 28 serine residues, only one, serine 195, was modified, resulting in a total loss of enzyme activity. This chemical modification reaction suggested that this unusually reactive serine residue plays a central role in the catalytic mechanism of chymotrypsin.

Figure 9.2

An Unusually Reactive Serine in Chymotrypsin. Chymotrypsin is inactivated by treatment with diisopropylphosphofluoridate (DIPF), which reacts only with serine 195 among 28 possible serine residues.

9.1.2 Chymotrypsin Action Proceeds in Two Steps Linked by a Covalently Bound Intermediate

How can we elucidate the role of serine 195 in chymotrypsin action? A study of the enzyme's kinetics provided a second clue to chymotrypsin's catalytic mechanism and the role of serine 195. The kinetics of enzyme action are often easily monitored by having the enzyme act on a substrate analog that forms a colored product. For chymotrypsin, such a chromogenic substrate is N-acetyl-l-phenylalanine p-nitrophenyl ester. This substrate is an ester rather than an amide, but many proteases will also hydrolyze esters. One of the products formed by chymotrypsin's cleavage of this substrate is p- nitrophenolate, which has a yellow color (Figure 9.3). Measurements of the absorbance of light revealed the amount of p-nitrophenolate being produced.

Figure 9.3

Chromogenic Substrate. N-Acetyl-l-phenylalanine p-nitrophenyl ester yields a yellow product, p-nitrophenolate, on cleavage by chymotrypsin. p-Nitrophenolate forms by deprotonation of p-nitrophenol at pH 7.

Under steady-state conditions, the cleavage of this substrate obeys Michaelis-Menten kinetics with a K_M of 20 μM and a k_cat of 77 s^-1. The initial phase of the reaction was examined by using the stopped-flow method. This technique permits the rapid mixing of enzyme and substrate and allows almost instantaneous monitoring of the reaction. At the beginning of the reaction, this method revealed a “burst” phase during which the colored product was produced rapidly (Figure 9.4). Product was then produced more slowly as the reaction reached the steady state. These results suggest that hydrolysis proceeds in two steps. The burst is observed because, for this substrate, the first step is more rapid than the second step.

Figure 9.4

Kinetics of Chymotrypsin Catalysis. Two stages are evident in the cleaving of N-acetyl-l-phenylalanine p-nitrophenyl ester by chymotrypsin: a rapid burst phase (pre-steady state) and a steady-state phase.

The two steps are explained by the reaction of the serine nucleophile with the substrate to form the covalently bound enzyme-substrate intermediate (Figure 9.5). First, the highly reactive serine 195 hydroxyl group attacks the carbonyl group of the substrate to form the acyl-enzyme intermediate, releasing the alcohol p-nitrophenol (or an amine if the substrate is an amide rather than an ester). Second, the acyl-enzyme intermediate is hydrolyzed to release the carboxylic acid component of the substrate and regenerate the free enzyme. Thus, p-nitrophenolate is produced rapidly on the addition of the substrate as the acyl-enzyme intermediate is formed, but it takes longer for the enzyme to be “reset” by the hydrolysis of the acyl-enzyme intermediate.

Figure 9.5

Covalent Catalysis. Hydrolysis by chymotrypsin takes place in two stages: (A) acylation to form the acyl-enzyme intermediate followed by (B) deacylation to regenerate the free enzyme.

9.1.3 Serine is Part of a Catalytic Triad That Also Includes Histidine and Aspartic Acid

The determination of the three-dimensional structure of chymotrypsin by David Blow in 1967 was a source of further insight into its mechanism of action. Overall, chymotrypsin is roughly spherical and comprises three polypeptide chains, linked by disulfide bonds. It is synthesized as a single polypeptide, termed chymotrypsinogen, which is activated by the proteolytic cleavage of the polypeptide to yield the three chains. The active site of chymotrypsin, marked by serine 195, lies in a cleft on the surface of the enzyme (Figure 9.6). The structural analysis revealed the chemical basis of the special reactivity of serine 195 (Figure 9.7). The side chain of serine 195 is hydrogen bonded to the imidazole ring of histidine 57. The -NH group of this imidazole ring is, in turn, hydrogen bonded to the carboxylate group of aspartate 102. This constellation of residues is referred to as the catalytic triad. How does this arrangement of residues lead to the high reactivity of serine 195? The histidine residue serves to position the serine side chain and to polarize its hydroxyl group. In doing so, the residue acts as a general base catalyst, a hydrogen ion acceptor, because the polarized hydroxyl group of the serine residue is poised for deprotonation. The withdrawal of the proton from the hydroxyl group generates an alkoxide ion, which is a much more powerful nucleophile than an alcohol is. The aspartate residue helps orient the histidine residue and make it a better proton acceptor through electrostatic effects.

Figure 9.6

Three-Dimensional Structure of Chymotrypsin. The three chains are shown in ribbon form in orange, blue, and green. The side chains of the catalytic triad residues, including serine 195, are shown as ball-and-stick representations, as are two intrastrand (more...)

Figure 9.7

The Catalytic Triad. The catalytic triad, shown on the left, converts serine 195 into a potent nucleophile, as illustrated on the right.

These observations suggest a mechanism for peptide hydrolysis (Figure 9.8). After substrate binding (step 1), the reaction begins with the hydroxyl group of serine 195 making a nucleophilic attack on the carbonyl carbon atom of the substrate (step 2). The nucleophilic attack changes the geometry around this carbon atom from trigonal planar to tetrahedral. The inherently unstable tetrahedral intermediate formed bears a formal negative charge on the oxygen atom derived from the carbonyl group. This charge is stabilized by interactions with NH groups from the protein in a site termed the oxyanion hole (Figure 9.9). These interactions also help stabilize the transition state that precedes the formation of the tetrahedral intermediate. This tetrahedral intermediate then collapses to generate the acyl-enzyme (step 3). This step is facilitated by the transfer of a proton from the positively charged histidine residue to the amino group formed by cleavage of the peptide bond. The amine component is now free to depart from the enzyme (step 4) and is replaced by a water molecule (step 5). The ester group of the acyl-enzyme is now hydrolyzed by a process that is essentially a repeat of steps 2 through 4. The water molecule attacks the carbonyl group while a proton is concomitantly removed by the histidine residue, which now acts as a general acid catalyst, forming a tetrahedral intermediate (step 6). This structure breaks down to form the carboxylic acid product (step 7). Finally, the release of the carboxylic acid product (step 8) readies the enzyme for another round of catalysis.

Figure 9.8

Peptide Hydrolysis by Chymotrypsin. The mechanism of peptide hydrolysis illustrates the principles of covalent and acid-base catalysis. The dashed green lines indicate favorable interactions between the negatively charged aspartate residue and the positively (more...)

Figure 9.9

The Oxyanion Hole. The structure stabilizes the tetrahedral intermediate of the chymotrypsin reaction. Hydrogen bonds (shown in green) link peptide NH groups and the negatively charged oxygen.

This mechanism accounts for all characteristics of chymotrypsin action except the observed preference for cleaving the peptide bonds just past residues with large, hydrophobic side chains. Examination of the threedimensional structure of chymotrypsin with substrate analogs and enzyme inhibitors revealed the presence of a deep, relatively hydrophobic pocket, called the S₁ pocket, into which the long, uncharged side chains of residues such as phenylalanine and tryptophan can fit. The binding of an appropriate side chain into this pocket positions the adjacent peptide bond into the active site for cleavage (Figure 9.10). The specificity of chymotrypsin depends almost entirely on which amino acid is directly on the amino-terminal side of the peptide bond to be cleaved. Other proteases have more-complex specificity patterns, as illustrated in Figure 9.11. Such enzymes have additional pockets on their surfaces for the recognition of other residues in the substrate. Residues on the amino-terminal side of the scissile bond (the bond to be cleaved) are labeled P₁, P₂, P₃, and so forth, indicating their positions in relation to the scissile bond. Likewise, residues on the carboxyl side of the scissile bond are labeled P₁′, P₂′, P₃′, and so forth. The corresponding sites on the enzyme are referred to as S₁, S₂ or S₁′, S₂′, and so forth.

Figure 9.10

The Hydrophobic Pocket of Chymotrypsin. The hydrophobic pocket of chymotrypsin is responsible for its substrate specificity. The key amino acids that constitute the binding site are labeled, including the active-site serine residue (boxed). The position (more...)

Figure 9.11

Specificity Nomenclature for Protease-Substrate Interactions. The potential sites of interaction of the substrate with the enzyme are designated P (shown in red), and corresponding binding sites on the enzyme are designated S. The scissile bond (also (more...)

9.1.4 Catalytic Triads Are Found in Other Hydrolytic Enzymes

Many other proteins have subsequently been found to contain catalytic triads similar to that discovered in chymotrypsin. Some, such as trypsin and elastase, are obvious homologs of chymotrypsin. The sequences of these proteins are approximately 40% identical with that of chymotrypsin, and their overall structures are nearly the same (Figure 9.12). These proteins operate by mechanisms identical with that of chymotrypsin. However, they have very different substrate specificities. Trypsin cleaves at the peptide bond after residues with long, positively charged side chains—namely, arginine and lysine—whereas elastase cleaves at the peptide bond after amino acids with small side chains—such as alanine and serine. Comparison of the S₁ pockets of these enzymes reveals the basis of the specificity. In trypsin, an aspartate residue (Asp 189) is present at the bottom of the S₁ pocket in place of a serine residue in chymotrypsin. The aspartate residue attracts and stabilizes a positively charged arginine or lysine residue in the substrate. In elastase, two residues at the top of the pocket in chymotrypsin and trypsin are replaced with valine (Val 190 and Val 216). These residues close off the mouth of the pocket so that only small side chains may enter (Figure 9.13).

Figure 9.12

Structural Similarity of Trypsin and Chymotrypsin. An overlay of the structure of chymotrypsin (red) on that of trypsin (blue) shows the high degree of similarity. Only α-carbon atom positions are shown. The mean deviation in position between (more...)

Figure 9.13

The S₁ Pockets of Chymotrypsin, Trypsin, and Elastase. Certain residues play key roles in determining the specificity of these enzymes. The side chains of these residues, as well as those of the active-site serine residues, are shown in color.

Other members of the chymotrypsin family include a collection of proteins that take part in blood clotting, to be discussed in Chapter 10. In addition, a wide range of proteases found in bacteria and viruses also belong to this clan. Furthermore, other enzymes that are not homologs of chymotrypsin have been found to contain very similar active sites. As noted in Chapter 7, the presence of very similar active sites in these different protein families is a consequence of convergent evolution. Subtilisin, a protease in bacteria such as Bacillus amyloliquefaciens, is a particularly well characterized example. The active site of this enzyme includes both the catalytic triad and the oxyanion hole. However, one of the NH groups that forms the oxyanion hole comes from the side chain of an asparagine residue rather than from the peptide backbone (Figure 9.14). Subtilisin is the founding member of another large family of proteases that includes representatives from Archaea, Eubacteria, and Eukarya.

Figure 9.14

The Catalytic Triad and Oxyanion Hole of Subtilisin. The peptide bond attacked by nucleophilic serine 221 of the catalytic triad will develop a negative charge, which is stabilized by enzyme NH groups (both in the backbone and in the side chain of Asn (more...)

Yet another example of the catalytic triad has been found in carboxypeptidase II from wheat. The structure of this enzyme is not significantly similar to either chymotrypsin or subtilisin (Figure 9.15). This protein is a member of an intriguing family of homologous proteins that includes esterases such as acetylcholine esterase and certain lipases. These enzymes all make use of histidine-activated nucleophiles, but the nucleophiles may be cysteine rather than serine. Finally, other proteases have been discovered that contain an active-site serine or threonine residue that is activated not by a histidine-aspartate pair but by a primary amino group from the side chain of lysine or by the N-terminal amino group of the polypeptide chain.

Figure 9.15

Carboxypeptidase II. The structure of carboxypeptidase II from wheat (right) is illustrated with its two chains (blue and red). The catalytic triad of carboxypeptidase II (left) is composed of the same amino acids as those in chymotrypsin, despite the (more...)

Thus, the catalytic triad in proteases has emerged at least three times in the course of evolution. We can conclude that this catalytic strategy must be an especially effective approach to the hydrolysis of peptides and related bonds.

9.1.5 The Catalytic Triad Has Been Dissected by Site-Directed Mutagenesis

The techniques of molecular biology discussed in Chapter 6 have permitted detailed examination of the catalytic triad. In particular, site-directed mutagenesis has been used to test the contribution of individual amino acid residues to the catalytic power of an enzyme. Subtilisin has been extensively studied by this method. Each of the residues within the catalytic triad, consisting of aspartic acid 32, histidine 64, and serine 221, has been individually converted into alanine, and the ability of each mutant enzyme to cleave a model substrate has been examined (Figure 9.16). As expected, the conversion of active-site serine 221 into alanine dramatically reduced catalytic power; the value of k_cat fell to less than one-millionth of its value for the wild-type enzyme. The value of K_M was essentially unchanged: its increase by no more than a factor of two indicated that substrate binding is not significantly affected. The mutation of histidine 64 to alanine had very similar effects. These observations support the notion that the serine-histidine pair act together to generate a nucleophile of sufficient power to attack the carbonyl group of a peptide bond. The conversion of aspartate 32 into alanine had a smaller effect, although the value of k_cat still fell to less than 0.005% of its wild-type value. The simultaneously conversion of all three catalytic triad residues into alanine was no more deleterious than the conversion of serine or histidine alone. Despite the reduction in their catalytic power, the mutated enzymes still hydrolyze peptides a thousand times as rapidly as does buffer at pH 8.6.

Figure 9.16

Site-Directed Mutagenesis of Subtilisin. Residues of the catalytic triad were mutated to alanine, and the activity of the mutated enzyme was measured. Mutations in any component of the catalytic triad cause a dramatic loss of enzyme activity. Note that (more...)

Because the oxyanion hole of subtilisin includes a side-chain NH group in addition to backbone NH groups, it is possible to probe the importance of the oxyanion hole for catalysis by site-directed mutagenesis. The mutation of asparagine 155 to glycine reduced the value of k_cat to 0.2% of its wild-type value but increased the value of K_M by only a factor of two. These observations demonstrate that the NH group of the asparagine residue plays a significant role in stabilizing the tetrahedral intermediate and the transition state leading to it.

9.1.6 Cysteine, Aspartyl, and Metalloproteases Are Other Major Classes of Peptide-Cleaving Enzymes

Not all proteases utilize strategies based on activated serine residues.

Classes of proteins have been discovered that employ three alternative approaches to peptide-bond hydrolysis (Figure 9.17). These classes are the (1) cysteine proteases, (2) aspartyl proteases, and (3) metalloproteases. In each case, the strategy generates a nucleophile that attacks the peptide carbonyl group (Figure 9.18).

Figure 9.17

Three Classes of Proteases and Their Active Sites. These examples of a cysteine protease, an aspartyl protease, and a metalloprotease use a histidine-activated cysteine residue, an aspartate-activated water molecule, and a metal-activated water molecule, (more...)

Figure 9.18

The Activation Strategies for Three Classes of Proteases. The peptide carbonyl group is attacked by (A) a histidine-activated cysteine, in the cysteine proteases; (B) an aspartate-activated water molecule, in the aspartyl proteases; and (C) a metalactivated (more...)

The strategy used by the cysteine proteases is most similar to that used by the chymotrypsin family. In these enzymes, a cysteine residue, activated by a histidine residue, plays the role of the nucleophile that attacks the peptide bond (see Figure 9.18), in a manner quite analogous to that of the serine residue in serine proteases. An ideal example of these proteins is papain, an enzyme purified from the fruit of the papaya. Mammalian proteases homologous to papain have been discovered, most notably the cathepsins, proteins having a role in the immune and other systems. The cysteine-based active site arose independently at least twice in the course of evolution; the caspases, enzymes that play a major role in apoptosis (Section 2.4.3), have active sites similar to that of papain, but their overall structures are unrelated.

The second class comprises the aspartyl proteases. The central feature of the active sites is a pair of aspartic acid residues that act together to allow a water molecule to attack the peptide bond. One aspartic acid residue (in its deprotonated form) activates the attacking water molecule by poising it for deprotonation, whereas the other aspartic acid residue (in its protonated form) polarizes the peptide carbonyl, increasing its susceptibility to attack (see Figure 9.18). Members of this class include renin, an enzyme having a role in the regulation of blood pressure, and the digestive enzyme pepsin. These proteins possess approximate twofold symmetry, suggesting that the two halves are evolutionarily related. A likely scenario is that two copies of a gene for the ancestral enzyme fused to form a single gene that encoded a single-chain enzyme. Each copy of the gene would have contributed an aspartate residue to the active site. The human immunodeficiency virus (HIV) and other retroviruses contain an unfused dimeric aspartyl protease that is similar to the fused protein, but the individual chains are not joined to make a single chain (Figure 9.19). This observation is consistent with the idea that the enzyme may have originally existed as separate subunits.

Figure 9.19

The Structure of HIV Protease and Its Binding Pocket. The protease is a dimer of identical subunits, shown in blue and yellow, consisting of 99 amino acids each. The active-site aspartic acid residues, one from each chain, are shown as ball-and-stick (more...)

The metalloproteases constitute the final major class of peptide-cleaving enzymes. The active site of such a protein contains a bound metal ion, almost always zinc, that activates a water molecule to act as a nucleophile to attack the peptide carbonyl group. The bacterial enzyme thermolysin and the digestive enzyme carboxypeptidase A are classic examples of the zinc proteases. Thermolysin, but not carboxypeptidase A, is a member of a large and diverse family of homologous zinc proteases that includes the matrix metalloproteases, enzymes that catalyze the reactions in tissue remodeling and degradation.

In each of these three classes of enzymes, the active site includes features that allow for the activation of water or another nucleophile as well as for the polarization of the peptide carbonyl group and subsequent stabilization of a tetrahedral intermediate (see Figure 9.18).

9.1.7 Protease Inhibitors Are Important Drugs

Compounds that block or modulate the activities of proteases can have dramatic biological effects. Most natural protease inhibitors are similar in structure to the peptide substrates of the enzyme that each inhibits (Section 10.5.4). Several important drugs are protease inhibitors. For example, captopril, an inhibitor of the metalloprotease angiotensin-converting enzyme (ACE), has been used to regulate blood pressure. Crixivan, an inhibitor of the HIV protease, is used in the treatment of AIDS. This protease cleaves multidomain viral proteins into their active forms; blocking this process completely prevents the virus from being infectious (see Figure 9.19). To prevent unwanted side effects, protease inhibitors used as drugs must be specific for one enzyme without inhibiting other proteins within the body.

Let us examine the interaction of Crixivan with HIV protease in more detail. Crixivan is constructed around an alcohol that mimics the tetrahedral intermediate; other groups are present to bind into the S₂, S₁, S₁′, and S₂′ recognition sites on the enzyme (Figure 9.20). The results of x-ray crystallographic studies revealed the structure of the enzyme-Crixivan complex, showing that Crixivan adopts a conformation that approximates the twofold symmetry of the enzyme (Figure 9.21). The active site of HIV protease is covered by two apparently flexible flaps that fold down on top of the bound inhibitor. The hydroxyl group of the central alcohol interacts with two aspartate residues of the active site, one in each subunit. In addition, two carbonyl groups of the inhibitor are hydrogen bonded to a water molecule (not shown), which, in turn, is hydrogen bonded to a peptide NH group in each of the flaps. This interaction of the inhibitor with water and the enzyme is not possible with cellular aspartyl proteases such as renin and thus may contribute to the specificity of Crixivan and other inhibitors for HIV protease.

Figure 9.20

Crixivan, an HIV Protease Inhibitor. The structure of Crixivan is shown in comparison with that of a peptide substrate of HIV protease. The scissile bond in the substrate is highlighted in red.

Figure 9.21

HIV Protease-Crixivan Complex. (Left) The HIV protease is shown with the inhibitor crixivan bound at the active site. (Right) The drug has been rotated to reveal its approximately twofold symmetric conformation.