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

ABSTRACT:Previous studies in our laboratory showed that among cDNAexpressed human cytochrome P450 (P450) supersomes, CYP2C19 was the most active in methoxychlor-O-demethylation (Hu et al., 2004). However, based on the 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 b 5 , and lipid, 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 depended on the amount of CPR and 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 their catalytic rates, which depend on the substrate undergoing metabolism.

Functional Interactions between Cytochromes P450 1A2 and 2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle

“…These complexes could be disrupted by an increase in the ionic strength of the buffer, suggesting that the interactions between the P450s were electrostatic in nature (23,24). Recently, a similar type of interaction also has been reported for CYP2C9 and CYP2C19 in which the activity of CYP2C9 is activated, whereas that of CYP2C19 is inhibited by the presence of the alternate enzyme (25). Thus, it seems that some P450-P450 interactions may have an important role in the regulation of enzyme activity.…”

“…Several studies have shown that the metabolism of numerous substrates by a given P450 enzyme is altered in the presence of other P450s (21,25,(42)(43)(44). The specific interaction with mixed reconstituted systems containing both CYP1A2 and FIGURE 4.…”

Global Analysis of Protein−Protein Interactions Reveals Multiple CYP2E1−Reductase Complexes

“…We did not consider the alternate pathways to generate complexes. Like in other studies (19,39), our system was at equilibrium and therefore (29) we could not even in principle determine the path through which these complexes were generated. Our equilibrium binding studies could only reveal which complexes were most likely to be present under reaction conditions.…”

“…Our initial titrations and Job plot were limited subsets of data for the system, and thus limited the ability to assess the role of protein-protein interactions in regulating P450 activity. Similarly, reliance on the titration format and small data sets may explain why groups were not able to actually fit data from mixed P450 systems to possible complexation mechanisms (19,32,39,40).…”

“…Our initial titrations and Job plot were limited subsets of data for the system, and thus limited the ability to assess the role of protein-protein interactions in regulating P450 activity. Similarly, reliance on the titration format and small data sets may explain why groups were not able to actually fit data from mixed P450 systems to possible complexation mechanisms (19,32,39,40).As discussed by Beechem (41), global analysis of multi-dimensional data, for example, where multiple component concentrations are simultaneously varied and the entire superset is analyzed as a whole, is always more informative about the underlying biochemical mechanism, in comparison with trying to analyze individual subsets of data separately. For this study, we generated a two-dimensional data set where both P450 and CPR were simultaneously varied over a wide range of concentrations.…”