Uncompetitive inhibition bisubstrate reaction

The modality of compounds that inhibit enzymes catalyzing bisubstrate reactions will differ with respect to the two substrates of the reaction, and the pattern of inhibition will depend on the reaction mechanism of the enzyme. Thus, when we use terms like competitive, noncompetitive, or uncompetitive inhibition, we must... 

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. 

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. 

Uncompetitive inhibition is rarely observed in single-substrate reactions but is frequently observed in multi substrate reactions. An uncompetitive inhibitor can provide information about the order of binding of the different substrates. In a bisubstrate-catalyzed reaction, for example, a given inhibitor may be competitive with respect to one of the two substrates and uncompetitive with respect to the other. The linear plots for classical uncompetitive inhibition patterns are described by Equation 17.19 and are illustrated in Fig. 17.8. 

As a mle, an uncompetitive inhibition occurs only if there are more than one substrate or product (Huang, 1990). For example, an uncompetitive inhibition will take place in a Rapid Equilibrium Order bisubstrate reaction, when an inhibitor competes with B while A is the variable substrate. Thus, the equilibria shown below describe an ordered bisubstrate system in which an inhibitor competes with B but does not bind to free enzyme. 

We shall turn now to much more realistic cases of bisubstrate reactions. The proper way to study a substrate inhibition in bisubstrate reactions is to vary a noninhibitory substrate, at differing high levels of the inhibitory one and see whether the slopes, intercepts, or both of reciprocal plots show the inhibitory effects (Cleland, 1979). These cases are then called competitive, uncompetitive, and noncompetitive substrate inhibition, respectively. 

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