Enzymes (Second Edition)

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inactivation of the enzyme requires two sequential steps: a binding event and an inactivation event. Irreversible inhibitors that behave in this fashion display a linear relationship between 1/k and 1/[I] that intersects the y axis at a value greater than zero (Figure 10.7B). If, however, the formation of the reversible EI complex is kinetically insignificant relative to the rate of inactivation, the double-reciprocal plot will pass through the origin, reflecting a single-step inactivation process (Kitz and Wilson, 1962):

E I k E I

Although not as common as the two-step inactivation scheme shown in Figure 10.1D, this type of behavior is sometimes seen for small molecule affinity labels of enzymes. For example, Kitz and Wilson (1962) showed that the compound methylsulfonyl fluoride inactivates acetylcholinesterease by irreversible formation of a sulfonyl—enzyme adduct that appears to form in a single inactivation step (Figure 10.8).

10.3 DISTINGUISHING BETWEEN MODES OF INHIBITOR INTERACTION WITH ENZYME

Morrison states that almost all slow binding enzyme inhibitors act as competitive inhibitors, binding at the enzyme active site (Morrison, 1982; Morrison and Walsh, 1988). Nevertheless it is possible, in principle at least, for slow binding inhibitors to interact with the enzyme by competitive, noncompetitive, or uncompetitive inhibition patterns. In the preceding equations, the relationships between K and K , and between K* and K*, are the same as those presented in Chapters 8 and 9 for the relationships between K and K for the different modes of inhibition.

To distinguish the mode of inhibition that is taking place, hence to ensure the use of the appropriate relationships for K and K* in the equations, one must determine the effects of varying substrate concentration on the value of k at a fixed concentration of inhibitor. Tian and Tsou (1982, and references therein) have presented derivations of the relationships between k and substrate concentration for competitive, noncompetitive, and uncompetitive irreversible inhibitors. (Similar patterns will be observed for slow binding inhibitors that conform to Scheme C as well.) More generalized forms of these relationships are given in Equations 10.15—10.17.

Figure 10.8 (A) Plot of kobs as a function of inhibitor concentration for inhibition of acetylcholinesterase by methylsulfonyl fluoride. (B) The data in (A) as a double-reciprocal plot. [Data adapted from Kitz and Wilson (1962).]

The constant k in these equations can be treated as an empirical variable for curve-fitting purposes (see Tian and Tsou, 1982, for the explicit form of k for irreversible inhibitors).

From the forms of Equations 10.15—10.17, we see that a competitive slow binding inhibitor will display a diminution in k as the substrate concentra-

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Figure 10.9 Expected dependence of kobs on substrate concentration for time-dependent irreversible inhibitors that conform to competitive (circles), noncompetitive (triangles), and uncompetitive (squares) modes of interaction with the enzyme.

tion is raised. For a noncompetitive inhibitor, on the other hand, k will not

vary with substrate concentration (when 1), while for an uncompetitive inhibitor the value of k will increase with increasing substrate concentration. These relationships between k and substrate concentration are illustrated in Figure 10.9.

10.4 DETERMINING REVERSIBILITY

It has been established that when an inhibitor conforming to Scheme C of Figure 10.1 has a very low value of k , it is difficult to differentiate this mode of inhibition from true irreversible inactivation according to Scheme D. To distinguish between these two possibilities, one must determine whether enzyme activity can be rescued by removal of unbound inhibitor from the enzyme solution. This is typically accomplished by large dilution, dialysis, filter binding, or size exclusion chromatography (see Chapters 4 and 7 and references therein for details about these methods). Suppose, for example, that a slow binding inhibitor reduces the steady state velocity of an enzyme reaction by 50% at a concentration of 100 nM. If we prepare a 1 mL sample of enzyme with inhibitor present at 100 nM and dialyze this sample extensively against a liter of buffer, the final concentration of enzyme in the dialysis tubing will be

essentially unchanged, but the concentration of free inhibitor will be reduced 1000-fold. If the inhibitor were binding reversibly to the enzyme, we would have observed a postdialysis return of enzyme activity to close to the original uninhibited activity. For a very low value of k , it might take some time— hours or days—for the new equilibrium between free and bound inhibitor to establish itself after dialysis. If k is nonzero, however, the expected reversal of inhibition eventually will occur. Of course, one must ensure that the enzyme itself is stable during these manipulations. Otherwise, it will be impossible to distinguish residual inhibition (due to the inhibitor) from enzyme inactivation (due to protein denaturation).

To distinguish covalent inactivation from noncovalent inhibition, one can look for the release of the original inhibitor molecule upon denaturation of the enzyme sample. Suppose that a slow binding inhibitor actually acted as a covalent affinity label of the target enzyme. If we were to denature the enzyme after inhibition and then separate the denatured protein from the rest of the solution (see Chapter 7), the inhibitory molecule would remain with the denatured protein, as a result of the covalent linkage between the inhibitor and the enzyme. If, on the other hand, the inhibitor were noncovalently associated with the enzyme, it would be released into the solution upon denaturation of the enzyme.

An illustration of this type of experiment comes from work performed in our laboratory on an inhibitor of the inducible isoform of the enzyme prostaglandin G/H synthase (PGHS2). A compound we were investigating, DuP697, displayed the kinetic features of a competitive, slow binding, irreversible enzyme inactivator (Copeland et al., 1994). A plot of k as a function of DuP697 concentration displayed a hyperbolic fit that passed through the origin of the plot. Extensive dialysis of the inhibited enzyme against buffer did not result in a return of enzymatic activity, suggesting either that the inhibitor was covalently associating with the enzyme or that the value of k was extremely small.

To determine which way DuP697 was interacting with the enzyme, we treated a micromolar solution of the enzyme with a substoichiometric concentration of the inhibitor and allowed the resulting solution to equilibrate for a long time period, relative to the rate of enzyme inactivation. The enzyme was then denatured and precipitated by addition of 4 volumes of a 1 : 1 mixture of methanol/acetonitrile. The denatured protein solution was centrifuged through a 30 kDa cutoff filter, and the filtrate from this treatment was dried under nitrogen and redissolved in a small volume of dimethyl sulfoxide or acetonitrile. The amount of DuP697 released from the enzyme by this treatment was then determined by reversed phase HPLC and by measuring the ability of the redissolved filtrate to effect inhibition of fresh samples of the enzyme (Copeland et al., 1994, 1995). Upon finding that 97% of the DuP697 added to the starting enzyme sample was recovered in this way, we concluded that DuP697 is not a covalent modifier of the enzyme, but rather conforms to Scheme C of Figure 10.1 with an extremely small value for k .

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10.5 EXAMPLES OF SLOW BINDING ENZYME INHIBITORS

The literature is filled with examples of slow binding, slow tight binding, affinity label, and mechanism-based inhibitors of important enzymes. Extensive examples of slow binding inhibitors were presented in the review by Morrison and Walsh (1988). Silverman has devoted a two-volume text to the subject of mechanism-based enzyme inactivators (1988a), as well as an extensive review article (1988b) of their potential uses in medicine. (See also Trzaskos et al., 1995, for an interesting more recent example of mechanism-based inactivation of lanosterol 14 -methyl demethylase in the design of new cholesterol-lowering therapies.) Rather than providing an exhaustive review of the literature, we shall present two examples of specific enzyme systems that have proved amenable to time-dependent inhibition: the serine proteases and prostaglandin G/H synthase. These examples should suffice to illustrate the importance of this general class of enzyme inhibitors. In Section 10.5.3 we also present a general discussion of irreversible affinity labels as mechanistic probes of enzyme structure and mechanism.

10.5.1 Serine Proteases

As we saw in Chapter 6, the serine proteases hydrolyze peptide bonds through the formation of a tetrahedral transition state involving a peptide carbonyl carbon of the substrate and an active site serine residue as the attacking nucleophile. Several groups have taken advantage of the ability of boron to adopt a tetrahedral ligand sphere in preparing transition state analogues as inhibitors of serine proteases. Kettner and Shervi (1984) have used this strategy to prepare selective inhibitors of the serine proteases chymotrypsin and leukocyte elastase based on -aminoboronate peptide analogues (Figure 10.10). They found that succinamide methyl esters that incorporate aminoboronate analogues of phenylalanine and valine were highly selective inhibitors of chymotrypsin and leukocyte elastase, respectively. A selective inhibitor of leukocyte elastase could have potential therapeutic value in the treatment of a number of inflammatory diseases of the respiratory system (e.g., cystic fibrosis, asthma). Kinetic studies of R-Pro-boroPhe-OH binding to chymotrypsin and R-Pro-boroVal-OH binding to leukocyte elastase revealed that both inhibitors function as competitive slow binding inhibitors that conform to Scheme C of Figure 10.1. For chymotrypsin inhibition by R-Pro-boroPhe-OH, these workers determined values of K and K* of 3.4 and 0.16 nM, respectively. Likewise, for leukocyte elastase inhibition by R-Pro-boroVal-OH, the values of K and K* were found to be 15 and 0.57 nM, respectively. Interestingly, Kettner and Shervi also found R-Pro-boroPhe-OH to be a nanomolar inhibitor of the serine protease cathepsin G, but in this case no slow binding behavior was observed. They suggest that the slow binding behavior of these inhibitors reflects the formation of an initial tetrahedral adduct with the active

Figure 10.10 Examples of slow binding -aminoboronate peptide inhibitors of serine proteases. These inhibitors form tetrahedral adducts with the active site serine of the proteases.

See Kettner and Shervi (1984) for further details.

site serine, followed by a conformational rearrangement of the enzyme to optimize binding (presumably this conformational readjustment does not occur in the case of cathepsin G).

10.5.2 Prostaglandin G/H Synthase

Prostaglandins are mediators of many of the physiological effects associated with inflammation that lead to such symptoms as pain, swelling, and fever. The biosynthesis of these mediators is rate-limited by the conversion of arachidonic acid to prostaglandin GH by the enzyme prostaglandin G/H synthase (PGHS). One of the most widely used drugs today for the treatment of the pain, swelling, and fever associated with inflammation is aspirin, a compound first isolated from the bark of a certain type of willow tree that had been used for centuries as a folk treatment for pain and fever (Weissman, 1991).

In the early 1970s Vane and his coworkers showed that aspirin elicits its anti-inflammatory effects by inhibition of prostaglandin biosynthesis (Vane,

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1971). It was subsequently found that aspirin functions as an affinity label for the enzyme PGHS, covalently inhibiting the enzyme by acetylating an active site serine (Ser 530). The acetylation of this residue irreversibly blocks the binding of arachidonic acid to the enzyme active site. Chronic aspirin use, however, may lead to stomach pain and ulceration, and renal failure, as a result of the breakdown of the mucosal linings of the stomach, intestines, and kidneys. For years, scientists and physicians have searched for anti-inflamma- tory drugs that could be taken over time without severe side effects. From their efforts a broad class of drugs, known as nonsteroidal anti-inflammatory drugs (NSAIDs) has emerged.

A large and highly prescribed class of NSAIDs are the carboxylic acid containing compounds, typified by the drugs flurbiprofen and indomethacin (Figure 10.11). These compounds have been shown to act as time-dependent inactivators of PGHS, conforming to Scheme C of Figure 10.1. However, the value of k is so low that for all practical purposes, these compounds function as irreversible inactivators and can be treated kinetically as such. Rome and Lands (1975), who have studied the time dependence of these inhibitors in detail, noted a common structural feature, a carboxylic acid group. Reasoning that some acid—base chemistry at the enzyme active site might account for the time-dependent inhibitory effects observed, these workers prepared the methyl esters of eight carboxylate-containing PGHS inhibitors. The results of these studies are summarized in Table 10.1.

Rome and Lands found that for the most part, binding of the inhibitor (reflected in K ) was not significantly affected by esterification, but in all cases the time dependence (reflected in k ) was completely lost.

A structural rationale for the foregoing results may now be available. Picot et al. (1994) have reported the crystal structure of PGHS with the carboxylate inhibitor flurbiprofen bound at the active site. They found that the carboxylate moiety of this inhibitor engages in formation of a salt bridge with Arg 120 in the arachidonic acid binding cavity. The formation of this salt bridge, by displacement of nearby amino acid residues, may be the rate-limiting step in the time-dependent inactivation of the enzyme by these inhibitors. Figure 10.11 provides a cartoon version of the proposed interactions between the active site arginine and the carboxylate group of NSAIDs. Note that residue Ser 530 is in close proximity to the bound inhibitor in the crystal structure. This serine is the site of covalent modification and irreversible inactivation by aspirin.

Recently Penning and coworkers (Tang et al., 1995) developed affinity labels of PGHS by preparing bromoacetamido analogues of the existing NSAIDs indomethacin and mefenamic acid. The bromoacetamido group is attacked by an active site nucleophile to form a covalent adduct that leads to irreversible inactivation of the enzyme (Figure 10.12). Under strong acidic conditions, the amine-containing NSAID moiety is cleaved off, leaving behind a carboxymethylated version of the active site nucleophile. These affinity labels can thus be used as mechanistic probes of the enzyme active site. By incorporating a radiolabel into the methylene carbon of the bromoacetamido group, one can

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Table 10.1 Time-dependent kinetic parameters for carboxylate inhibitors of PGHS and their methyl esters

Source: Data from Rome and Lands (1975).

obtain selective radiolabel incorporation into the enzyme at the attacking nucleophile.

PGHS performs two catalytic conversions of its substrate, arachidonic acid: a cyclooxygenase step (in which two equivalents of molecular oxygen are added) and a peroxidase step (in which the incorporated peroxide moiety is converted to the final alcohol of prostaglandin GH ). The classical NSAID inhibitors block enzyme turnover by inhibiting selectively the cyclooxygenase step of the reaction. This observation has raised the question of whether the two enzymatic reaction steps involve the same set of active site residues or use distinct catalytic centers for each reaction. Tang et al. (1995) demonstrated that the bromoacetamido affinity labels bind to PGHS in a 2 : 1 stoichiometry and, unlike their NSAID analogues, abolish both the cyclooxygenase and peroxidase activities of the enzyme. Interestingly, they found that pretreatment of the enzyme with aspirin or mefenamic acid reduces the stoichiometry of the affinity label incorporation to 1 : 1. Furthermore, if the mefenamic acid saturated enzyme is treated with the affinity label and subsequently dialyzed to remove mefenamic acid, the version of the enzyme that results retains its cyclooxygenase activity but is devoid of peroxidase activity (Tang et al., 1995). These findings support the hypothesis that the catalytic centers for cyclooxygenase and peroxidase activities are distinct in PGHS. These affinity labels, and the peroxidase-deficient enzyme they provide, should prove to be useful tools in dissecting the mechanism of PGHS turnover.

A continuing problem in the treatment of inflammatory diseases with NSAIDs is the gastrointestinal and renal damage observed among patients who receive chronic treatment with these drugs. The side effects are mechan- ism-based in that inhibition of PGHS not only blocks the symptoms of inflammation but also interferes with the maintenance of the protective mucosal linings of the digestive system. In the early 1990s it was discovered

Figure 10.12 Affinity labeling of PGHS by the bromoacetamido analogue of the NSAID 2,3-dimethylanthranilic acid. [Adapted from Tang et al. (1995).]

that humans and other mammals contain two isoforms of this enzyme: PGHS1, which is constitutively expressed in a wide variety of tissues, including gastrointestinal and renal tissue, and PGHS2, which is induced in response to inflammatory stimuli and is primarily localized to cells of the immune system and the brain. This discovery immediately suggested a mechanism for treating inflammatory disease without triggering the side effects of traditional NSAID therapy, namely, selective inhibition of the inducible isoform, PGHS2.

In the hope of developing new and safer anti-inflammatory drugs, a number of laboratories, including ours, set out to identify compounds that would inhibit PGHS2 selectively over PGHS1. One compound that seemed to fit this selectivity profile was DuP697, a methylsulfonyl-containing diaryl thiophene (Figure 10.13). Kinetic studies of DuP697 inhibition of PGHS1 and PGHS2 revealed an unusual basis for the isozyme selectivity of this compound. DuP697 appeared to bind weakly, but with equal affinity, to both isozymes (Copeland et al., 1994). For PGHS1, we demonstrated that DuP697 acted as a classic reversible competitive inhibitor (Copeland et al., 1995). For PGHS2, however, the binding of DuP697 induced an isomerization of the enzyme that led to much tighter association of the inhibitor—enzyme complex, according to Scheme C of Figure 10.1. This isomerization step in fact led to such tight binding that the inhibition could be treated as a two-step irreversible inactivation of the enzyme (Scheme D of Figure 10.1). Plots of k as a function of DuP697 concentration showed the hyperbolic behavior expected for inactivation where k was zero or near zero. From this we determined values of K and

kof 2.19 M and 0.017 s , respectively, or a second-order rate constant

/K of 7.76 10 M · s . Thus the isozyme selectivity of this compound resulted from its ability to induce a conformational transition in one

isozyme but not the other. The structural basis for this inhibitor-induced conformational transition remains to be fully elucidated.

We then explored analogues of DuP697 in an attempt to identify the minimal structural requirements for selective PGHS2 inhibition and to search