Inhibitor uncompetitive

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. 

As stated above, the vast majority of slow binding inhibitors that have been reported in the literature are active-site directed, hence competitive inhibitors. Nevertheless, there is no theoretical reason why noncompetitive or uncompetitive inhibitors could not also display slow binding behavior. Thus, to convert the apparent values of K,... 

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). 

An uncompetitive inhibitor binds to the ES complex rather than the free ens me. 

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. 

Figure 8.8 The kinetic effects of an uncompetitive inhibitor. The effects of an uncompetitive inhibitor are very complex and the reaction usually shows a parallel decrease in both the maximum velocity and the Michaelis constant.

In addition to the Lineweaver-Burk plot (see p.92), the Eadie-Hofstee plot is also commonly used. In this case, the velocity v is plotted against v /[A]. In this type of plot, Vmax corresponds to the intersection of the approximation lines with the v axis, while Km is derived from the gradient of the lines. Competitive and non-competitive inhibitors are also easily distinguishable in the Eadie-Hofstee plot. As mentioned earlier, competitive inhibitors only influence Km, and not Vmax- The lines obtained in the absence and presence of an inhibitor therefore intersect on the ordinate. Non-competitive inhibitors produce lines that have the same slope (llower level. Another type of inhibitor, not shown here, in which Vmax and lselective binding of the inhibitor to the EA complex. 

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. 

D. L. Purich H. J. Fromm (1972) Biochem. Biophys. Acta, 268, 1. Note Uncompetitive inhibitors display parallel lines in Dixon plots. 

If a noncompetitive or an uncompetitive inhibitor were present with the substrate at constant ratio, then graphical analysis would suggest that the phenomenon of substrate inhibition is present. If an investigator analyzed the apparent substrate inhibition via a Marmasse plot, wrong estimates of both the K a and K s values would be reported and the investigator would be mislead with respect to the kinetic mechanism. If partial inhibitors or alternative substrates are present in constant ratio, depending on the relative sizes of the Ymax and values,... 

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 inhibition can also be a possibility in multisubstrate reactions. For example, in an ordered Bi Bi reaction, a competitive inhibitor with respect to the second substrate B, will act as an uncompetitive inhibitor with respect to the first substrate, A. 

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. 

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