Enzymes uncompetitive inhibitors

Reversible inhibition of an enzyme is competitive, uncompetitive, or mixed. Competitive inhibitors compete with substrate by binding reversibly to the active site, but they are not transformed by the enzyme. Uncompetitive inhibitors bind only to the ES complex, at a site distinct from the active site. Mixed inhibitors bind to either E or ES, again at a site distinct from the active site. In irreversible inhibition an inhibitor binds permanently to an active site by forming a covalent bond or a veiy stable noncovalent interaction. 

Most inhibition of enzymes is competitive, uncompetitive, or noncompetitive. Competitive inhibitors reversibly compete with substrate for the same site on free enzyme. Uncompetitive inhibitors bind only to the enzyme-substrate complex and not the free enzyme. Noncompetitive inhibitors can bind to both the enzyme and the enzyme-substrate complex. 

Like a noncompetitive inhibitor, an uncompetitive inhibitor does not compete with the substrate since it binds to the enzyme—substrate complex but not to the free enzyme. Uncompetitive inhibition... 

An inhibitor that binds exclusively to the ES complex, or a subsequent species, with little or no affinity for the free enzyme is referred to as uncompetitive. Inhibitors of this modality require the prior formation of the ES complex for binding and inhibition. Hence these inhibitors affect the steps in catalysis subsequent to initial substrate binding that is, they affect the ES —> ES1 step. One might then expect that these inhibitors would exclusively affect the apparent value of Vm and not influence the value of KM. This, however, is incorrect. Recall, as illustrated in Figure 3.1, that the formation of the ESI ternary complex represents a thermodynamic cycle between the ES, El, and ESI states. Hence the augmentation of the affinity of an uncompetitive inhibitor that accompanies ES complex formation must be balanced by an equal augmentation of substrate affinity for the El complex. The result of this is that the apparent values of both Vmax and Ku decrease with increasing concentrations of an uncompetitive inhibitor (Table 3.3). The velocity equation for uncompetitive inhibition is as follows ... 

Figure 3.12 Substrate titration of steady state velocity for an enzyme in the presence of an uncompetitive inhibitor at varying concentrations. (A) Untransformed data (B) data as in (A) plotted on a semilog scale (C) data as in (A) plotted in double reciprocal form. For all three plots the data are fit to Equation (3.6).

In this chapter we described the thermodynamics of enzyme-inhibitor interactions and defined three potential modes of reversible binding of inhibitors to enzyme molecules. Competitive inhibitors bind to the free enzyme form in direct competition with substrate molecules. Noncompetitive inhibitors bind to both the free enzyme and to the ES complex or subsequent enzyme forms that are populated during catalysis. Uncompetitive inhibitors bind exclusively to the ES complex or to subsequent enzyme forms. We saw that one can distinguish among these inhibition modes by their effects on the apparent values of the steady state kinetic parameters Umax, Km, and VmdX/KM. We further saw that for bisubstrate reactions, the inhibition modality depends on the reaction mechanism used by the enzyme. Finally, we described how one may use the dissociation constant for inhibition (Kh o.K or both) to best evaluate the relative affinity of different inhibitors for ones target enzyme, and thus drive compound optimization through medicinal chemistry efforts. 

We saw in Chapter 3 that bisubstrate reactions can conform to a number of different reaction mechanisms. We saw further that the apparent value of a substrate Km (KT) can vary with the degree of saturation of the other substrate of the reaction, in different ways depending on the mechanistic details. Hence the determination of balanced conditions for screening of an enzyme that catalyzes a bisubstrate reaction will require a prior knowledge of reaction mechanism. This places a necessary, but often overlooked, burden on the scientist to determine the reaction mechanism of the enzyme before finalizing assay conditions for HTS purposes. The importance of this mechanistic information cannot be overstated. We have already seen, in the examples of methotrexate inhibition of dihydrofolate, mycophenolic acid inhibiton of IMP dehydrogenase, and epristeride inhibition of steroid 5a-reductase (Chapter 3), how the [5]/A p ratio can influence one s ability to identify uncompetitive inhibitors of bisubstrate reactions. We have also seen that our ability to discover uncompetitive inhibitors of such reactions must be balanced with our ability to discover competitive inhibitors as well. 

At very low substrate concentration ([S] approaches zero), the enzyme is mostly present as E. Since an uncompetitive inhibitor does not combine with E, the inhibitor has no effect on the velocity and no effect on Vmsa/Km (the slope of the double-reciprocal plot). In this case, termed uncompetitive, the slopes of the double-reciprocal plots are independent of inhibitor concentration and only the intercepts are affected. A series of parallel lines results when different inhibitor concentrations are used. This type of inhibition is often observed for enzymes that catalyze the reaction between two substrates. Often an inhibitor that is competitive against one of the substrates is found to give uncompetitive inhibition when the other substrate is varied. The inhibitor does combine at the active site but does not prevent the binding of one of the substrates (and vice versa). 

Not all inhibitors fall into either of these two classes but some show much more complex effects. An uncompetitive inhibitor is defined as one that results in a parallel decrease in the maximum velocity and the Km value (Figure 8.8). The basic mode of action of such an inhibitor is to bind only to the enzyme-substrate complex and not to the free enzyme and so it reduces the rate of formation of products. Alkaline phosphatase (EC 3.1.3.1) extracted from rat intestine is inhibited by L-phenylalanine in such a manner. 

Full and partial uncompetitive inhibitory mechanisms, (a) Reaction scheme for full uncompetitive inhibition indicates ordered binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor prevents release of product, (b) Lineweaver-Burk plot for full uncompetitive inhibition reveals a series of parallel lines and an increase in the 1/v axis intercept to infinity at infinitely high inhibitor concentrations. In this example, Ki = 3 iulM. (c) Replot of Lineweaver-Burk slopes from (b) is linear, confirming a full inhibitory mechanism, (d) Reaction scheme for partial uncompetitive inhibition indicates random binding of substrate and inhibitor to two mutually exclusive sites. The presence of inhibitor alters the rate of release of product (by a factor P) and the affinity of enzyme for substrate (by a factor a) to an identical degree, while the presence of substrate alters the affinity of enzyme for inhibitor by a. (e) Lineweaver-Burk plot for partial uncompetitive inhibition reveals a series of parallel lines and an increase in the 1/v axis intercept to a finite value at infinitely high inhibitor concentrations. In this example, Ki = 3 iulM and a = = 0.5. (f) Replot of Lineweaver-Burk slopes from (e) is hyperbolic, confirming a partial inhibitory mechanism... 

Rule 1. Upon obtaining a double-reciprocal plot of 1/v vx. 1/[A] (where [A] is the initial substrate concentration and V is the initial velocity) at varying concentrations of the inhibitor (I), if the vertical intercept varies with the concentration of the reversible inhibitor, then the inhibitor can bind to an enzyme form that does not bind the varied substrate. For example, for the simple Uni Uni mechanism (E + A EX E -P P), a noncompetitive or uncompetitive inhibitor (both of which exhibit changes in the vertical intercept at varying concentrations of the inhibitor), I binds to EX, a form of the enzyme that does not bind free A. In such cases, saturation with the varied substrate will not completely reverse the inhibition. 

Inhibition of an enzyme-catalyzed reaction in which the inhibitor does not bind to the free, uncomplexed enzyme and does not compete with the substrate for the enzyme s active site . Eor a Uni Uni mechanism (E -t A EX E + P), an uncompetitive inhibitor would bind to... 

However, note that is replaced with Xia (the dissociation constant of A for the free enzyme) in the rapid-equilibrium equation. A standard double-reciprocal plot (1/v v. 1/[A]) at different concentrations of inhibitor will yield a series of parallel lines. A vertical intercept v. [I] secondary replot will provide a value for X on the horizontal axis. If questions arise as to whether the lines are truly parallel, one possibility is to replot the data via a Hanes plot ([A]/v v. [A]). In such a plot, the lines of an uncompetitive inhibitor intersect on the vertical axis. 

Uncompetitive inhibitors can bind to the enzyme-substrate complex only, but not to the free enzyme molecule. The Lineweaver-Burk plots in such cases give parallel straight lines for activity-substrate concentration profiles, measured at different concentrations of the inhibitor (Figure 8.4), according to equation ... 

Uncompetitive inhibitors. These bind only to the enzyme-snbstrate complex, not to free enzyme. This results in a decrease in the maximnm rate of reaction and means that less enzyme is available to bind snbstrate. 

FIGURE 6-15 Three types of reversible inhibition, (a) Competitive inhibitors bind to the enzyme s active site, (b) Uncompetitive inhibitors bind at a separate site, blit bind only to the ES complex. K, is the equilibrium constant for inhibitor binding to E K is the equilibrium constant for inhibitor binding to ES. (c) Mixed inhibitors bind at a separate site, but may bind to either E or ES. 

Two other types of reversible inhibition, uncompetitive and mixed, though often defined in terms of one-substrate enzymes, are in practice observed only with enzymes having two or more substrates. An uncompetitive inhibitor (Fig. 6-15b) binds at a site distinct from the substrate active site and, unlike a competitive inhibitor, binds only to the ES complex. In the presence of an uncompetitive inhibitor, the Michaelis-Menten equation is altered to... 

In practice, uncompetitive and mixed inhibition are observed only for enzymes with two or more substrates—say, Sj and S2—and are very important in the experimental analysis of such enzymes. If an inhibitor binds to the site normally occupied by it may act as a competitive inhibitor in experiments in which [SJ is varied. If an inhibitor binds to the site normally occupied by S2, it may act as a mixed or uncompetitive inhibitor of Si. The actual inhibition patterns observed depend on whether the and S2-binding events are ordered or random, and thus the order in which substrates bind and products leave the active site can be determined. Use of one of the reaction products as an inhibitor is often particularly informative. If only one of two reaction products is present, no reverse reaction can take place. However, a product generally binds to some part of the active site, thus serving as an inhibitor. Enzymologists can use elaborate kinetic studies involving different combinations and amounts of products and inhibitors to develop a detailed picture of the mechanism of a bisubstrate reaction. 

So far the only well-characterized uncompetitive inhibitor is L-phenyl-alanine, shown to be uncompetitive for intestinal (180) and placental phosphatases (42). By contrast, D-phenylalanine has no inhibitory effect. The L isomer apparently acts by preventing the breakdown of phosphoryl phosphatase (170), possibly by blocking the acceptor site mentioned in Section III,D,4. A list of concentrations required to produce 50% inhibition of a wide variety of phosphatases showed that all the enzymes were affected though there was a spread in susceptibilities from 0.8 mM for HeLa cell culture phosphatase to 26 mM for mouse intestinal phosphatase... 

Uncompetitive inhibitors bind only to the enzyme-substrate complex and not to the free enzyme. For example, the substrate binds to the enzyme causing a conformational change which reveals the inhibitor binding site, or it could bind directly to the enzyme-bound substrate. In neither case does the enzyme compete for the same binding site, so the inhibition cannot be overcome by increasing the substrate concentration. Scheme 5.A5.2 below illustrates this uncompetitive behaviour. 

Competitive Inhibitors Bind at the Active Site Noncompetitive and Uncompetitive Inhibitors Do Not Compete Directly with Substrate Binding Irreversible Inhibitors Permanently Alter the Enzyme Structure... 

Types of enzyme inhibition, (a) A competitive inhibitor competes with the substrate for binding at the same site on the enzyme, (b) A noncompetitive inhibitor binds to a different site but blocks the conversion of the substrate to products, (c) An uncompetitive inhibitor binds only to the enzyme—substrate complex. (E = enzyme S = substrate.)... 

Still another possibility is that the inhibitor binds only to the enzyme-substrate complex and not to the free enzyme (fig. 7.14c). This reaction is called uncompetitive inhibition. Uncompetitive inhibition is rare in reactions that involve a single substrate but more common in reactions with multiple substrates. Plots of 1/v versus 1/[S] at different concentrations of an uncompetitive inhibitor give a series of parallel lines. 

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