Bisubstrate

Such a reaction is termed a bisubstrate reaction. In general, bisubstrate reactions proceed by one of two possible routes ... 

FIGURE 14.18 Single-displacement bisubstrate mechanism. Double-reciprocal plots of the rates observed with different fixed concentrations of one substrate (B here) are graphed versus a series of concentrations of A. Note that, in these Lineweaver-Burk plots for singledisplacement bisubstrate mechanisms, the lines intersect to the left of the 1/v axis. 

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. 

Bisubstrate analogs are compounds that contain features of both substrates for an enzymatic reaction in which two substrates are used. 

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-... 

Table 3.6 Pattern of dead-end inhibition observed for bisubstrate reactions...

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. 

The determination of bisubstrate reaction mechanism is based on a combination of steady state and, possibly, pre-steady state kinetic studies. This can include determination of apparent substrate cooperativity, as described in Chapter 2, study of product and dead-end inhibiton patterns (Chapter 2), and attempts to identify... 

If we are able to covalently link these two compounds together in a way that captures all their individual binding interactions, the free energy of binding for the bisubstrate inhibitor will be the sum of the free energies of the individual molecules that is, the free energy, assuming no loses of productive interactions, will be -14.4 kcal/mol. The Kt of this combined, bisubstrate inhibitor will therefore be... 

A recent example of the success of the bisubstrate, transition state design approach comes from the work of Pope and coworkers (Pope et al., 1998a-c Brown et al., 2000) on the design of inhibitors of bacterial isoleucyl tRNA synthetase... 

Figure 7.10 Chemical structure of SB-234764, a tight binding bisubstrate inhibitor of bacterial isoleucine tRNA synthetase.

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... 

For bisubstrate reactions that conform to a ternary complex mechanism (see Chapter 3), inactivation should require the presence of the noncognate substrate. 

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.

Over the past decade, however, the PTK inhibitors favored by most investigators have been ATP mimics that compete with ATP at the binding site. Most of the compounds depicted in Table 2 are ATP mimics with the exception of the tyrphostins developed by us. In the case of tyrphostins one can indeed classify compounds which compete with ATP, compounds which compete with the substrate and bisubstrate inhibitors which compete with the substrate and ATP simultaneously [18]. Compounds can also be identified that act as mixed competitive inhibitors which bind simultaneously with ATP and/or substrate but decrease the affinity of ATP and the substrate to their respective sites [18,22]. Among the tyrphostins all classes of compounds can be identified [18] but the real question is which of these is preferable for clinical development. 

However, bisubstrate inhibitors incorporating a farnesyl- and a CAAX mimetic are very promising since one can expect that they display enhanced... 

---