SUBSTRATE-DEPENDENT COMPETITION OF DIFFERENT P450 ISOZYMES FOR LIMITING NADPH-CYTOCHROME P450 REDUCTASE

The goal of these studies was to demonstrate that one P450 isozyme can influence the function of another isozyme when combined in a reconsti tuted system. Benzphetamine and 7-pentoxyresorufin were both shown to be preferred substrates for P450 2B4 (LM2) as compared to P450 1A2 (LM 4). However, these substrates exhibited different characteristics when examined in reconstituted systems containing reductase and both P450 isozymes. Whereas benzphetamine demethylation showed a small increase in catalytic activity when both P450 1A2 and 2B4 were present over the activities obtained in simple reconstituted systems, 7-pentoxyresoruf in O-dealkylation (PROD) was dramatically inhibited when both isozymes were present. These results indicate that the functional interactions between P450s in complex reconstituted systems are dependent on the s ubstrate present. Inhibition of PROD was also dependent on reductase l evels, with the inhibitory effect being more pronounced at subsaturati ng reductase. Finally, these protein-protein interactions were shown t o be dependent on the reductase concentration in the reconstituted sys tem rather the P450 concentration, supporting the view that P450 1A2 i s inhibiting the reaction by competing with P450 2B4 for reductase mol ecules.