Alternative inhibitor detection

Although the pathway of Eq. (1) is now based on much evidence (Section 111) and is unambiguous in the case of at least one bacterium [Pseudomonas stutzeri strain Zobell (f. sp. P. perfectomarina)], there have been alternative hypothesis. One hypothesis, advanced by the Hollocher group (Garber and Hollocher, 1981 St. John and Hollocher, 1977), considered NO as a likely intermediate, but one that remained at least partly enzyme-bound and was not entirely free to diffuse. This view was based on the outcome of certain kinetic and isotope experiments which can be summarized as follows. When denitrifying bacteria were challenged simultaneously with [ N]nitrite and ordinary NO, the cells reduced both compounds concomitantly to N2 (or to N2O in the presence of acetylene which is a specific inhibitor (Balderston et al., 1976 Yoshinari and Knowles, 1976) of nitrous oxide reductase). In the process, little NO was generally detected in the gas phase pool of NO and there was relatively little isotopically mixed N2O formed. That is, most of the N and N reduced to NjO appeared as N2O... 

It should be noted that the mechanism depicted in Scheme 1 is the simplest that is consistent with mechanism-based inhibition. The mechanism for a given inhibitor and enzyme may be considerably more complex due to (a) multiple intermediates [e.g., MIC formation often involves four or more intermediates (29)], (b) detectable metabolite that may be produced from more than one intermediate, and (c) the fact that enzyme-inhibitor complex may produce a metabolite that is mechanistically unrelated to the inactivation pathway. Events such as these will necessitate alternate definitions for Z inact, Kh and r in terms of the microrate constants of the appropriate model. The hyperbolic relationship between rate of inactivation and inhibitor concentration will, however, remain, unless nonhyperbolic kinetics characterize this interaction. Silverman discussed this possibility from the perspective of an allosteric interaction between inhibitor and enzyme (5). Nonhyperbolic kinetics has been observed for the interaction of several drugs with members of the CYPs (30). 

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