Rapid, reversible enzyme inhibitors

Binding of a reversible inhibitor to an enzyme is rapidly reversible and thus bound and unbound enzymes are in equilibrium. Binding of the inhibitor can be to the active site, or to a cofactor, or to some other site on the protein leading to allosteric inhibition of enzyme activity. The degree of inhibition caused by a reversible inhibitor is not time-dependent the final level of inhibition is reached almost instantaneously, on addition of inhibitor to an enzyme or enzyme-substrate mixture. 

Herbicidal Inhibition of Enzymes. The Hst of known en2yme inhibitors contains five principal categories group-specific reagents substrate or ground-state analogues, ie, rapidly reversible inhibitors affinity and photo-affinity labels suicide substrate, or inhibitors and transition-state, or reaction-intermediate, analogues, ie, slowly reversible inhibitors (106). 

Regulation of enzyme activity is achieved in a variety of ways, ranging from controls over the amount of enzyme protein produced by the cell to more rapid, reversible interactions of the enzyme with metabolic inhibitors and activators. Chapter 15 is devoted to discussions of enzyme regulation. Because most enzymes are proteins, we can anticipate that the functional attributes of enzymes are due to the remarkable versatility found in protein structures. 

The inactivation is normally a first-order process, provided that the inhibitor is in large excess over the enzyme and is not depleted by spontaneous or enzyme-catalyzed side-reactions. The observed rate-constant for loss of activity in the presence of inhibitor at concentration [I] follows Michaelis-Menten kinetics and is given by kj(obs) = ki(max) [I]/(Ki + [1]), where Kj is the dissociation constant of an initially formed, non-covalent, enzyme-inhibitor complex which is converted into the covalent reaction product with the rate constant kj(max). For rapidly reacting inhibitors, it may not be possible to work at inhibitor concentrations near Kj. In this case, only the second-order rate-constant kj(max)/Kj can be obtained from the experiment. Evidence for a reaction of the inhibitor at the active site can be obtained from protection experiments with substrate [S] or a reversible, competitive inhibitor [I(rev)]. In the presence of these compounds, the inactivation rate Kj(obs) should be diminished by an increase of Kj by the factor (1 + [S]/K, ) or (1 + [I(rev)]/I (rev)). From the dependence of kj(obs) on the inhibitor concentration [I] in the presence of a protecting agent, it may sometimes be possible to determine Kj for inhibitors that react too rapidly in the accessible range of concentration. ... 

Figure 5.9 Recovery of enzyme activity after rapid dilution as described in Figure 5.8. Curve a represents the expected behavior for a control sample that was pre-incubated and diluted in the absence of inhibitor. Curve b represents the expected behavior for a rapidly reversible inhibitor. Curve c represents the expected behavior for a slowly reversible inhibitor, and curve d represents the expected behavior for an irreversible or very slowly reversible inhibitor. See color insert.

If the inhibition is found to be rapidly reversible, we must next determine if the approach to equilibrium for the enzyme-inhibitor complex is also rapid. As described in Chapter 4, some inhibitors bind slowly to their target enzymes, on a time scale that is long in comparision to the time scale of the reaction velocity measurement. The effect of such slow binding inhibition is to convert the linear progress curve seen in the absence of inhibitor to a curvilinear function (Figure 5.10). When nonlinear progress curves are observed in the presence of inhibitor, the analysis of... 

Carbamates are used as insecticides, nematocides, fungicides, and herbicides the toxicity of carbamate insecticides is similar to that of OP compounds and is based on the inhibition of ACHE. Also, carbamate metabolites may inhibit ACHE but are usually weaker inhibitors than the unchanged compound. Cholinesterase inhibition caused by carbamates is labile, of short duration, and rapidly reversible in fact, the half-life of the inhibited enzymes ranges between some minutes and 2 to 3 hours for RBC-ACHE and is on the order of some minutes for PCHE. Accumulation of cholinesterase activity on repeated exposures, as observed with OP compounds, does not occur with... 

The kinetics of the general enzyme-catalyzed reaction (equation 10.1-1) may be simple or complex, depending upon the enzyme and substrate concentrations, the presence/absence of inhibitors and/or cofactors, and upon temperature, shear, ionic strength, and pH. The simplest form of the rate law for enzyme reactions was proposed by Henri (1902), and a mechanism was proposed by Michaelis and Menten (1913), which was later extended by Briggs and Haldane (1925). The mechanism is usually referred to as the Michaelis-Menten mechanism or model. It is a two-step mechanism, the first step being a rapid, reversible formation of an enzyme-substrate complex, ES, followed by a slow, rate-determining decomposition step to form the product and reproduce the enzyme ... 

A (rapidly) reversible inhibitor will permit rapid and complete recovery of enzyme activity by dialysis. However, irreversible inhibitors are not removed by this procedure. Recovery from tight-binding inhibition is usually slow it is not uncommon for several dialysis bags containing enzyme to be prepared and for activity in each to be determined at various time points following the commencement of dialysis. The off-rate of these inhibitors is generally more rapid at higher temperatures. 

B. Ritonavir is a potent inhibitor of CYP3A4, the enzyme that rapidly inactivates lopinavir. This combination includes a low dose of ritonavir that is not likely to cause serious side effects but instead inhibits lopinavir metabolism. Ritonavir and lopinavir are HIV protease inhibitors and do not affect reverse transcriptase. Lopinavir is almost completely eliminated by metabolism to inactive metabolites little is eliminated unchanged by the kidney. Lopinavir is not known to inhibit the ability of HIV to mutate. Lopinavir inhibits the enzyme HIV protease, not a structural protein. 

Reversible inhibition occurs rapidly in a system which is near its equilibrium point and its extent is dependent on the concentration of enzyme, inhibitor and substrate. It remains constant over the period when the initial reaction velocity studies are performed. In contrast, irreversible inhibition may increase with time. In simple single-substrate enzyme-catalysed reactions there are three main types of inhibition patterns involving reactions following the Michaelis-Menten equation competitive, uncompetitive and non-competitive inhibition. Competitive inhibition occurs when the inhibitor directly competes with the substrate in forming the enzyme complex. Uncompetitive inhibition involves the interaction of the inhibitor with only the enzyme-substrate complex, while non-competitive inhibition occurs when the inhibitor binds to either the enzyme or the enzyme-substrate complex without affecting the binding of the substrate. The kinetic modifications of the Michaelis-Menten equation associated with the various types of inhibition are shown below. The derivation of these equations is shown in Appendix S.S. 

Allosteric effectors are inhibitors or activators that bind to enzymes at sites distinct from the active sites. Allosteric regulation allows cells to adjust enzyme activities rapidly and reversibly in response to changes in the concentrations of substances that are structurally unrelated to the substrates or products. The initial steps in a biosynthetic pathway commonly are inhibited by the end products of the pathway, and numerous enzymes are regulated by ATP, ADP, or AMP. 

Classical NSAIDs and COX-2 inhibitors are time-dependent, irreversible inhibitors of hCOX-2, which is consistent with a two-step process, involving an initial rapid equilibrium binding of enzyme and inhibitor, followed by a slow formation of a tightly bound enzyme-inhibitor complex. COX-2 inhibitors show a time-independent inhibition of hCOX-1, consistent with the formation of a reversible enzyme-inhibitor complex (Ouellet and Percival 1995 Riendeau et al. 2001). 

The free enzyme (E) binds the substrate (S) to form a noncovalent enzyme-inhibitor complex (E. S). This is assumed to be a rapid, reversible process, not involving any chemical changes, and with the affinity of the substrate for the enzyme s active site being determined by the binding forces discussed above. A chemical transformation of substrate to product (P), initially in complex with enzyme (E. P), then takes place. Finally, the product (P) is released into the medium with concomitant regeneration of free enzyme (E). 

This class of inhibitors acts by binding to the target enzyme s active site in a rapid, reversible, and noncovalent fashion. The net result is that the active site is blocked and the substrate is prevented from binding. Accordingly, in designing inhibitors of this type, optimization of the noncovalent binding forces between the inhibitor and the active site of the enzyme is of paramount importance. 

L Irreversible inactivation. Inactivation by affinity labels leads to irreversible covalent bond formation between the enzyme and the inhibitor. Unlike the complex between and enzyme and a rapid, reversible inhibitor, the covalent enzyme-inhibitor complex is no longer in equilibrium with free enzyme and inhibitor. Therefore, exhaustive dialysis or gel filtration of the covalent enzyme-inhibitor complex cannot lead to the recovery of free, active enzyme. However, such experiments do not allow distinction among tight-binding, noncovalent inhibitors, affinity labels, and mechanism-based inactivators. 

Then in the 1980s there was a reappraisal of the benefit-to-harm balance of the MAO inhibitors. This spawned both a search for safer and more selective or rapidly reversible enzyme inhibitors (including moclobemide, toloxatone, and brofaromine), as well as a review and retrial of the older compounds. 

Ret er.stble inhibition, in contrast with irreversible inhibition, is acterized by a rapid dissociation of the enzyme-inhibitor complex. In the type of reversible inhibition called competitive inhibition, an enzyme can bind substrate (forming an ES complex) or inhibitor 1) but not both (ESI). The competitive inhibitor often resembles the substrate and binds to the active site of the enzyme (Figure 8.15). The substrate is thereby prevented from binding to the same active site. A competitive inhibitor dimmishes the rate oj catalysis by reducing the pro-por/ion of enzyme molecules bound to a substrate. At any given inhibitor concentration, competitive inhibition can be relieved by increasing... 

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