[5] Reversible enzyme inhibitors as mechanistic probes

Publisher Summary Although competitive inhibitors have been used extensively for a variety of reasons in enzyme experiments, their value as tools, for making a choice of kinetic mechanism, from among possible alternatives, was not realized until 1962, when Fromm and Zewe suggested that competitive inhibitors of substrates could be used to differentiate between the random and ordered mechanisms. Furthermore, in the latter case, a determination of the substrate binding order could be made from such experiments. This protocol is quite likely to be the simplest approach for differentiating between ordered and random Bi Bi possibilities. In addition, it has the advantage of permitting the kineticist to come to definitive conclusions from the studies of reactions in a single direction only. Its obvious limitation involves the requirement that a competitive inhibitor be available for each substrate. However, when other initial rate data are available, a good deal of information, concerning the kinetic mechanism, may be provided from the experiments with only one substrate analog even for Bi and Ter reactant systems. In this chapter, it is assumed that the competitive inhibitor, which is usually a substrate analog, when bound to the enzyme, will not permit product formation to occur. These inhibitors are then dead-end inhibitors as contrasted with “partial” competitive inhibitors that when associated with the enzyme allow the formation of product either at a reduced or accelerated rate. In addition, it is assumed here that enzyme-inhibitor complex formation occurs rapidly relative to other steps in the reaction pathway.