EVALUATION OF CYTOCHROME-P450 MECHANISM AND KINETICS USING KINETIC DEUTERIUM-ISOTOPE EFFECTS

In this paper two hypotheses are tested: (i) the active oxygen species is similar in energetics for all cytochrome P450 (CYP) enzymes and (i i) linear free-energy relationships can be used to evaluate the mechan ism of the reaction of these enzymes. A series of intramolecular isoto pe effects were determined and compared for CYPs 1A2, 2B1, 2C9, 2E1, a nd P450cam. The results indicate that the isotope effects are very sim ilar for each of these isoforms of P450 and that the first hypothesis is likely to be true. Attempts to establish a linear free-energy relat ionship were only moderately successful: log V-max = 0.11 sigma(p)(+) + 1.73; r(2) = 0.588. It was determined, through the use of intermolec ular isotope effects, that the rates of hydrogen atom abstraction are masked. Thus, the second hypothesis is found to be false. This is like ly to be a general result for CYP reactions, and linear free-energy re lationships can only be used to determine the mechanism under very spe cial circumstances. In all cases, the rate-limiting step should be eva luated with isotope effect experiments before any mechanistic conclusi ons can be drawn. If the intermolecular isotope effects are found to b e masked, no mechanistic conclusion can be drawn from the linear free- energy relationship study.