Mechanism-Based Enzyme Inactivation Kinetics

Mechanism-based enzyme inactivation kinetics refers to the irreversible inhibition of an enzyme via a catalytically formed reactive intermediate that binds covalently (typically) to the enzyme active site prior to release and causes permanent inactivation of the enzyme. This type of inhibition is also known as 

Silverman (1998) has defined seven criteria that must be satisfied in order for a process to be characterized as involving mechanism-based inactivation. These are time dependence, saturation, substrate protection, irreversibility, stoichiometry of inactivation, involvement of a catalytic step, and inactivation occurs prior to release of the activated species. This review will not expound on each 

FIGURE 4.11 Plots representative of the decreasing enzyme activity with respect to the time and concentration dependence of mechanism-based inactivation kinetics. 

FIGURE 4.12 Kitz-Wilson plot of the half-lives (rate constants) for mechanism-based inactivation at each inactivator concentration. 

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In order for a compound to be defined as a mechanism-based enzyme inactivator, its reaction with the target enzyme must meet a simple set of kinetic and mechanistic criteria. The kinetics of the inactivation reaction should conform to those expected for the minimal scheme shown in Eq. (1). 

Fig. I. (a) Typical lime-dependent inactivation kinetics for a mechanism-based enzyme inactivator. (b) Replot of inactivation kinetics.

As a first step in determining the kinetics of mechanism-based enzyme inactivation, the time-dependence of the reaction is typically studied. In these experiments, the effects of preincubation (primary incubation) with increasing concentrations of the inactivator over various time intervals is studied for their effects on catalytic activity of the enzyme toward the reporter substrate. This typically results in a plot similar to that represented in Figure 4.11. 

Mechanism-based CYP inhibition or irreversible inhibition, involves permanent inactivation of CYP enzymes during catalysis, where reactive intermediate(s) are formed, leading to apoprotein or heme-ion center modification. Typical characteristics of mechanism-based enzyme inhibition include time-dependent loss of enzyme activity, a rate of inactivation generally following saturation kinetics, enzyme activity that cannot be recovered after... 

SCHEME 163 Proposed kinetic scheme for mechanism-based enzyme inhibition. E, I and P stand for enzyme, inhibitor and produet, respectively, and El is the initial binding of inhibitor to enzyme (the enzyme-inhibitor complex) and El is the aetive form of the complex in whieh the inhibitor is catalyzed to intermediate (reactive metabolite). Ejnact is the inactivated enzyme by the reactive metabolite formed. Inaetivation of the enzyme is an irreversible proeess over the time scale of the experiment. At the given eoneentrations of inhibitor and enzyme, the reactions are governed by the first-order rate eonstants ki, k i, k2,13 and I4, respeetively. 

In the kinetic scheme of Figure 8.2C, we see that once the active species is formed, it can go on to inactivate the enzyme directly or be released into solution. If the active species formed is a good affinity label (i.e., is highly electrophilic), there is a chance that this species will rebind and inactivate the enzyme as an affinity label. To be classified as a mechanism-based inactivator, the active species must be demonstrated to directly inactivate the enzyme while still bound, without reliance on dissociation from the EA complex. 

Linear furanocoumarins (psoralens) inhibit P450s as mechanism-based inactivators (suicide inhibitors). Thus, species that produce psoralens may have evolved C4H enzymes with enhanced tolerance to these compounds. Recombinant C4H from the psoralen-producing species R. graveolens showed markedly slower inhibition kinetics with psoralens, and possibly biologically significant tolerance, compared to C4H from a species that does not produce the compounds (H. tuberosus) ... 

Mechanism-based inhibition should be irreversible. Dialysis, ultrafiltration, or washing the protein (e.g., by isolating microsomes by centrifugation and resuspending them in drug-free buffer) will not restore enzyme activity, and the inhibition is highly resistant to sample dilution. Mechanism-based inhibition should be saturable. The rate of inactivation is proportional to the concentration of the inactivator until all enzyme molecules are saturated, in accordance with Michaelis-Menten kinetics. Additionally, the decrease in enzymatic activity over time should follow pseudo-first-order kinetics. 

It should be noted that the mechanism depicted in Scheme 1 is the simplest that is consistent with mechanism-based inhibition. The mechanism for a given inhibitor and enzyme may be considerably more complex due to (a) multiple intermediates [e.g., MIC formation often involves four or more intermediates (29)], (b) detectable metabolite that may be produced from more than one intermediate, and (c) the fact that enzyme-inhibitor complex may produce a metabolite that is mechanistically unrelated to the inactivation pathway. Events such as these will necessitate alternate definitions for Z inact, Kh and r in terms of the microrate constants of the appropriate model. The hyperbolic relationship between rate of inactivation and inhibitor concentration will, however, remain, unless nonhyperbolic kinetics characterize this interaction. Silverman discussed this possibility from the perspective of an allosteric interaction between inhibitor and enzyme (5). Nonhyperbolic kinetics has been observed for the interaction of several drugs with members of the CYPs (30). 

A mechanism-based inhibitor may be defined as a chemically unreactive compound that is treated by the target enzyme as a substrate, but instead of forming the usual product, it is converted into a highly reactive species via the normal catalytic mechanism. Prior to release from the active site, the reactive intermediate may alkylate amino acid functional groups, forming a new covalent bond and inactivating the enzyme (90). Irreversible, mechanism-based inactivation is typified by first-order, time-dependent loss of enzyme activity saturation kinetics inactivation protection by substrates and reversible inhibitors failure to recover activity following dialysis and usually a chemical stoichiometry of one covalent adduct formed per enzyme active site. 

One class of mechanism-based MAO inhibitors includes the unsaturated alkylamines (propargylamine analogs) (Table II). Although the kinetics of enzyme inactivation for these compounds are consistent with a mechanism-based inhibitor, in only a few cases has the chemical mechanism and site of protein modification been determined. Pargyline (iV-benzyl-N-methyl-2-propynylamine) is a classic example. Pargyline reacts stoichiometrically and irreversibly with the MAO of bovine kidney, with protection from inactivation afforded by substrate benzylamine (91). Furthermore, the reaction involves bleaching of the FAD cofactor at 455 nm and the formation of a new absorbing species at 410 nm and a covalent adduct of inactivator with flavin cofactor (92). 

A summary of some recently discovered inactivators of E. coli PFL is presented in Table V. Consistent with these compounds acting as mechanism-based or active site-directed inhibitors is the observation of pseudo-first-order inactivation kinetics, substrate protection (by pyruvate or formate for inhibitors that are pyruvate or formate analogs, respectively), and isotope effects on the rates of inactivation by the deuterated analogs. The details of some of these studies, the proposed inactivation mechanisms, and the implications to the normal enzymic reaction are discussed below. 

The crucial distinction between mechanism-based inactivators and other classes of enzyme inhibitors is the requirement for the catalytic action of the target enzyme to unmask the reactive functionality of the inactivator. Because the kinetics of inactivation by mechanism-based and affinity label approaches are identical, other methods must be used to demonstrate the necessity of enzyme action. The cofactors and additional substrates compulsory for turnover of normal substrates should be essential for the inactivation reaction, and the stereochemical constraints required of substrates should also be reflected in inactivator struc-... 

Hexanal phenylhydrazone also serves as an inactivator of soybean lipoxygenase 1 (L-1) in a process which demonstrates kinetics more complex than those of standard mechanism-based inactivators (Galey et al., 1988). Aerobic incubation of hexanal phenylhydrazone with L-1 leads to enzyme inactivation and conversion of the compound to its corresponding a-azo hydroperoxide, which is also an inactivator. Four equivalents of the a-azo hydroperoxide are sufficient to inactivate the enzyme completely, whereas the amount of the parent phenylhydrazone required to fiilly inactivate the enzyme increases from 13 to a maximum of 70 as the ratio of hexanal phenylhydrazone to L-1 increases. Since the partition ratio is normally independent of inhibitor and enzyme concentrations, a more complex mechanism is apparent. The addition to reaction mixtures of glutathione peroxidase, which reduces the a-azo hydroperoxide metabolite to the corresponding alcohol, suppresses about 80% of the inactivation. The a-azo hydro-... 

In contrast, 5-benzyl-6-chloro-2-pyrone is not a substrate for chymotrypsin, but inactivates the enzyme with the formation of a shoulder in the absorbance spectrum at 320 nm (Westkaemper and Abeles, 1983). The chromophore, which is similar to that of the pyrone ring, appears with kinetics corresponding to the rate of inactivation and is lost on reactivation of the enzyme. The crystal structure and C NMR studies of the modified enzyme demonstrate that the benzyl group is bound in the specificity pocket of the active site and that serine-195 is covalently attached to C-6 of the intact pyrone (Ringe et ai, 1985) (Fig. 49). Inactivation by the 5-benzyI analog is therefore an example of affinity labeling rather than mechanism-based inactivation and emphasizes the importance of the side chain of protease inactivators in proper orientation of the compound in the active site. 

Because L-vinylglycine also irreversibly inactivates ACC synthase in a time-dependent manner when the enzyme is incubated with L-vinylglycine and pyridoxal phosphate in the absence of AdoMet and because ACC synthase, like other pyridoxal-dependent enzymes, may catalyse p.-y-elimination of AdoMet to produce L-vinylglycine, it has been proposed that the mechanism-based inactivation proceeds through the formation of a vinylglycine-enzyme complex which is inactive (Fig. 3). Detailed kinetic studies indicate that the... 

CYP2C19 inactivation suggests that it occurs within the active site of the enzyme. The inactivation follows classical mechanism-based inactivation criteria with the following kinetic parameters i/2max = 3-4 min, = 3.2 X lO s , K, = 97 p-M, = 37 L mol s, and partition... 

Mechanisms of CYP inhibition can be broadly divided into two categories reversible inhibition and mechanism-based inactivation. Depending on the mode of interaction between CYP enzymes and inhibitors, reversible CYP inhibition is further characterized as competitive, noncompetitive, uncompetitive, and mixed (Ito et al., 1998b). Evaluation of reversible inhibition of CYP reactions is often conducted under conditions where M-M kinetics is obeyed. Based on the scheme illustrated in Fig. 5.1, various types of reversible inhibition are summarized in Table 5.1. Figure 5.1 depicts a simple substrate-enzyme complex during catalysis. In the presence of a reversible inhibitor, such a complex can be disrupted leading to enzyme inhibition. 

Kinetic model for mechanism-based inhibition is proposed in Scheme 16.3 (Waley, 1980 Walsh et al., 1978). Inactivation of the enzyme is an irreversible process over the time scale of the experiment. At the given concentrations of inhibitor and enzyme, the reactions indicated in Scheme 16.3 are governed by the first-order rate constants k, k, 2, k, and 4, respectively. The rate of enzyme inactivation can be introduced by Equation 16.3 (Jxmg and Metcalf, 1975 Kitz and Wilson, 1962). 

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