INTERPRETATIONS OF CYTOCHROME-P450 MECHANISMS FROM KINETIC-STUDIES

The catalytic mechanism of cytochrome P450 (P450) enzymes has generall y been understood in terms of a classic cycle in which electron donati on is often limiting and catalysis is understood in terms of hydrogen abstraction and rapid oxygen rebound. In the course of detailed invest igations with kinetic hydrogen isotope effects we have studied two sys tems in which somewhat unusual isotope effects have been interpreted i n terms of modifications of the general paradigm. The low isotope effe cts observed for N-demethylation reactions are in contrast to high val ues seen with P450-catalyzed C-hydroxylation and peroxidase-catalyzed N-demethylation and are consonant with a role for the P450 FeO2+ entit y in base-catalyzed deprotonation of an aminium radical. With P450 2E1 , kinetic deuterium isotope effects are seen on the apparent K-m for t he substrate (increased) but not on V-max. The results are interpreted in terms of a mechanism where C-H bond cleavage is sensitive to deute rium substitution but a step following this is rate-limiting. This ste p may be product release.