Bisubstrate enzyme mechanisms

Fromm and Rudolph have discussed the practical limitations on interpreting product inhibition experiments. The table below illustrates the distinctive kinetic patterns observed with bisubstrate enzymes in the absence or presence of abortive complex formation. It should also be noted that the random mechanisms in this table (and in similar tables in other texts) are usually for rapid equilibrium random mechanism schemes. Steady-state random mechanisms will contain squared terms in the product concentrations in the overall rate expression. The presence of these terms would predict nonhnearity in product inhibition studies. This nonlin-earity might not be obvious under standard initial rate protocols, but products that would be competitive in rapid equilibrium systems might appear to be noncompetitive in steady-state random schemes , depending on the relative magnitude of those squared terms. See Abortive Complex... 

Let us consider a more complicated mechanism, an Ordered Bi Bi mechanism which is very common with bisubstrate enzymes. In this mechanism, an enzyme reacts with two substrates in an ordered fashion affording two products, which are also released in an ordered fashion. 

In the literature, literally dozens of kinetic mechanisms have been proposed for bisubstrate enzymes (Alberty, 1958 Alberty Hammes, 1958 Teller Alberty, 1959 Wong Hanes, 1962 Fromm, 1967 Dalziel, 1969 Hurst, 1969 Rudolph Fromm, 1969, 1971, 1973). However, only those pathways that are either weU documented, or seem to be a logical extension of established mechanisms, will be presented in this and the following chapters. Thus, we shall divide the rapid equilibrium bisubstrate reactions into the following major types, according to the type and number of enzyme-substrate or enzyme-product complexes that can form (Alberty, 1953 Cleland, 1970, 1977 Fromm, 1979 Engel, 1996 Purich Allison, 2000) ... 

In contrast to class I flavin reductases, class II flavin reductases are bona fide flavoproteins producing a characteristic flavin-absorption spectrum and containing a bound flavin prosthetic group involved in the electron transfer from NAD(P)H to the flavin substrate. The reaction sequence for class II enzymes, as exemplified by the flavoprotein component of sulfite reductase from E. coli, follows a ping pong bisubstrate-biproduct mechanism (85). In this process, the NAD(P)H and flavin bind to the same site sequentially electrons are first transferred from NADPH to FAD and then from FAD to another flavin. 

Many other multisubstrate examples abound in metabolism. In effect, these situations are managed by realizing that the interaction of the enzyme with its many substrates can be treated as a series of uni- or bisubstrate steps in a multi-step reaction pathway. Thus, the complex mechanism of a multisubstrate reaction is resolved into a sequence of steps, each of which obeys the single- and double-displacement patterns just discussed. 

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... 

The example of methotrexate points out that the inhibition modality of dead end inhibitors, with respect to a specific substrate, will depend on the reaction mechanism of the target enzyme. Thus a complete understanding of inhibition mechanism requires an understanding of the underlying reaction mechanism of the target enzyme. A comprehensive discussion of these issues has been provided by Segel (1975). Table 3.6 summarizes the pattern of dead-end inhibition observed for competitive inhibitors of one substrate in the common bisubstrate reaction mecha-... 

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. 

Determining balanced conditions for a single substrate enzyme reaction is usually straightforward one simply performs a substrate titration of reaction velocity, as described in Chapter 2, and sets the substrate concentration at the thus determined Ku value. For bisubstrate and more complex reaction mechanism, however, the determination of balanced conditions can be more complicated. 

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. 

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... 

Figure 8.18 Mechanism-based inactivators of steroid 5a-reductase. (A) Finasteride, (B) the bisubstrate analogue formed by reaction of NADP1 with finasteride catalyzed by the enzyme, and (C) dutasteride.

Almost all enzymes—in contrast to the simplified description given on p. 92—have more than one substrate or product. On the other hand, it is rare for more than two substrates to be bound simultaneously. In bisubstrate reactions of the type A + B C+D, a number of reaction sequences are possible. In addition to the sequential mechanisms (see p.90), in which all substrates are bound in a specific sequence before the product is released, there are also mechanisms in which the first substrate A is bound and immediately cleaved. A part of this substrate remains bound to the enzyme, and is then transferred to the second substrate B after the first product C has been released. This is known as the ping-pong mechanism, and it is used by transaminases, for example (see p.l78). In the Lineweaver— Burk plot (right see p.92), it can be recognized in the parallel shifting of the lines when [B] is varied. 

Let us now examine the behavior of enzymes operating by way of ordered and random kinetic bisubstrate mechanisms ... 

FIGURE 6-13 Common mechanisms for enzyme-catalyzed bisubstrate reactions, (a) The enzyme and both substrates come together to form a ternary complex. In ordered binding, substrate 1 must bind before substrate 2 can bind productively. In random binding, the substrates can bind in either order. 

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. 

In the sequential mechanism, all substrates must bind to the enzyme before any product is released. Consequently, in a bisubstrate reaction, a ternary complex of the enzyme and both substrates forms. Sequential mechanisms are of two... 

While developed as possible therapeutics, bisubstrate analogs have found great utility in the dissection and characterization of enzyme structure and mechanism. As discussed below, bisubstrate analogs have been used extensively in structural studies, where the use of natural substrates would result in catalysis, to investigate the architecture of the active site at the Michaelis complex, and to define structural changes at the active site produced by allosteric effectors. In some cases, bisubstrate analogs that are formed during the reaction (a type of mechanism-based inhibitor) can help to support or eliminate proposed chemical mechanisms. 

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