Polymorphic variants of CYP2C9: Mechanisms involved in reduced catalytic activity

CYP2C9 catalyzes the demethylation of the biphasic kinetics substrate (S)-naproxen, and the CYP2C9*2 (R144C) and CYP2C9*3 (I359L) variants are associated with lower rates of (S)-naproxen demethylation. To assess the reasons for these reductions in catalytic activity of the two variants and potential substrate concentration-dependent differences in a biphasic kinetics substrate, cytochrome P450 (P450) cycle coupling and uncoupling were monitored during coincubation of (S)-naproxen and CYP2C9 over a range of P450 reductase concentrations. Coupling was greatest in the CYP2C9.1 enzyme, followed by CYP2C9.2, and then CYP2C9.3. Uncoupling in CYP2C9.1 and CYP2C9.3 was primarily to H2O2. In contrast, CYP2C9.2 uncoupled to excess water preferentially. The conversion of enzyme to the high spin state was similar in CYP2C9.1 and CYP2C9.2, but lower in CYP2C9.3. It is noteworthy that neither altered substrate binding nor altered interaction with reductase seemed to be involved in reduced catalysis. These results suggest that in addition to coupling differences, differential uncoupling to shunt products and differences in spin state help explain the reduced catalytic activity in these enzymes.