Received for publication August 4, 2004.
Revised October 7, 2004.
Accepted for publication October 8, 2004.

Interactions between CYP2C9 and CYP2C19 in Reconstituted Binary Systems Influence their Catalytic Activity: Possible rationale for the inability of CYP2C19 to catalyze Methoxychlor demethylation in human liver microsomes.

Abstract

Previous studies in our laboratory showed that among cDNA-expressed human P450 supersomes, CYP2C19 was the most active in methoxychlor-O-demethylation (Hu et al., 2004). However, based on lack of inhibition of methoxychlor-O-demethylation by monoclonal anti-CYP2C19 antibodies in human liver microsomes (HLM), CYP2C19 did not seem to catalyze that reaction in HLM. By contrast CYP2C9, much less active than CYP2C19 in supersomes, was the most active in HLM. The current study examines whether the lack of methoxychlor-O-demethylation by CYP2C19 in HLM was due to CYP2C19 exhibiting inferior competition for the NADPH-cytochrome-P450 reductase (CPR) versus CYP2C9, and explores the interactions between CYP2C9 and CYP2C19 in a singular and binary complex of a reconstituted system. When reconstituted with CPR, cytochrome b5 and lipid, both purified CYP2C19 and CYP2C9 catalyzed methoxychlor-O-demethylation. However, whereas equimolar CPR to CYP2C9 supported maximal rates of methoxychlor demethylation and diclofenac hydroxylation, the rate of methoxychlor demethylation by CYP2C19 was not fully saturated even with a 9-fold molar excess of CPR over CYP2C19. This behavior of CYP2C19 was also observed with S-mephenytoin as the substrate. When a binary reconstitution system was prepared by mixing CYP2C9 and CYP2C19 enzymes, methoxychlor-O-demethylation and S-mephenytoin hydroxylation by CYP2C19 were dramatically inhibited. Inhibition was dependent on the amount of CPR and on the substrate used. By contrast, in the incubation containing CYP2C9, diclofenac-hydroxylation was activated by the presence of CYP2C19. These results show that interactions among P450 enzymes can modulate each other's activity by affecting their catalytic rates that depend on the substrate undergoing metabolism.

Key words: CYP2C, cytochrome b5, cytochrome P450 catalyzed oxidations, cytochrome P450 function, halogenated hydrocarbons, insecticides, liver microsomes, monooxygenases, NADPH cytochrome P450 reductase

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