Binding uncompetitive

Figure 7.7 Plot of IC50 as a function of substrate concentration (plotted as the ratio [S]/ATM on the x-axis) for tight binding competitive (closed circles) and tight binding uncompetitive (open circles) enzyme inhibitors.

And for tight binding uncompetitive inhibition, the relationship is given by... 

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... 

P-site ligands inhibit adenylyl cyclases by a noncompetitive, dead-end- (post-transition-state) mechanism (cf. Fig. 6). Typically this is observed when reactions are conducted with Mn2+ or Mg2+ on forskolin- or hormone-activated adenylyl cyclases. However, under- some circumstances, uncompetitive inhibition has been noted. This is typically observed with enzyme that has been stably activated with GTPyS, with Mg2+ as cation. That this is the mechanism of P-site inhibition was most clearly demonstrated with expressed chimeric adenylyl cyclase studied by the reverse reaction. Under these conditions, inhibition by 2 -d-3 -AMP was competitive with cAMP. That is, the P-site is not a site per se, but rather an enzyme configuration and these ligands bind to the post-transition-state configuration from which product has left, but before the enzyme cycles to accept new substrate. Consequently, as post-transition-state inhibitors, P-site ligands are remarkably potent and specific inhibitors of adenylyl cyclases and have been used in many studies of tissue and cell function to suppress cAMP formation. 

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 ... 

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. 

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,... 

Figure 6.9 Effect of substrate concentration (relative to KM) on the value of kAs at a fixed concentration of a slow binding inhibitor that is competitive (closed circles), uncompetitive (open circles), or noncompetitive (a = 1, closed squares) with respect to the varied substrate.

Because mechanism-based inactivators behave as alternative substrates for the enzyme, they must bind in the enzyme active site. Binding of a mechanism-based inactivator is therefore mutually exclusive with binding of the cognate substrate of the normal enzymatic reaction (we say cognate substrate here because for bisubstrate reactions, the mechanism-based inactivator could be competitive with one substrate and noncompetitive or uncompetitive with the other substrate of the reaction, depending on the details of the reaction mechanism). Thus, as the substrate concentration is increased, the observed rate of inactivation should decrease (Figure 8.10) as... 

A few natural products, mostly polyphenols and fatty acids, are low micromolar inhibitors of Fabl. Based on a different template, (E)-oroidin (33) displays an uncompetitive binding mechanism, similar to the one observed for triclosan [54]. 

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). 

Uncompetitive inhibition is extremely rare in nature, and can arise when the inhibitor binds to the enzyme-substrate complex, rather than to the free enzyme, as in competitive inhibition [111]. In uncompetitive inhibition, an increase in the concentration of the inhibitor requires a disproportionately large increase in the concentration of the substrate to maintain the same metabolic turnover. 

The inhibition can be interpreted as an increase of the Michaelis constant KM. In the case of uncompetitive inhibition (Fig. 9B), the binding of the substrate to the enzyme is not affected. However, the [ES] complex becomes inactive upon binding of the inhibitor Using Kj —> oo, the corresponding rate equation is... 

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. 

Full uncompetitive inhibition (O Figure 4-lIa) occurs as a result of inhibitor binding (only) to the ES complex binding is thus ordered. It occurs rarely in unireactant systems but is a common inhibitory mechaitism in multireactant systems. Since ESI is nonproductive, high inhibitor concentrations can drive... 

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... 

Henderson has also described a graphical method which can distinguish between, and quantify the effects of, tight-binding inhibitors acting through competitive, noncompetitive, and uncompetitive mechanisms (Henderson, 1972). The practicalities of this method are discussed by Segel (1993). 

These equilibrium-binding relationships give rise to four different kinetic responses competitive inhibition, uncompetitive inhibition, non-competitive inhibition, mixed inhibition. Details of the kinetics of these types of inhibition and how dissociation constants for the reactions can be measured are provided in Appendix 3.6. 

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. 

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