THE R144C CHANGE IN THE CYP2C9-ASTERISK-2 ALLELE ALTERS INTERACTION OF THE CYTOCHROME-P450 WITH NADPH-CYTOCHROME P450 OXIDOREDUCTASE

We have examined the kinetics of substrate metabolism by cDNA-expresse d human CYP2C9 and the R144C variant, Both enzymes exhibited similar a pparent K-m values for (S)-warfarin 7-hydroxylation, diclofenac 4'-hyd roxylation and lauric acid 11-hydroxylation, In contrast, the R144C va riant (relative to CYP2C9) had slower rates of metabolism for all thre e substrates. The difference was most pronounced for (S)-warfarin. Sur prisingly, the magnitude of the difference was found to be dependent o n the cytochrome P450 to NADPH-cytochrome P450 reductase (OR) ratio in the system (the difference being more pronounced at higher OR to P450 ratios) implying that the R144C change affects interaction of the P45 0 with OR, The rates of (S)-warfarin 7-hydroxylation by CYP2C9 and the R144C variant also exhibited differential dependence on salt concentr ation which further supported a difference in interaction with OR, Whe n OR was bypassed and the hydroxylation was supported by cumene hydrop eroxide, no difference in the rates of diclofenac 4'-hydroxylation was observed for CYP2C9 and the R144C variant regardless of OR to P450 ra tio, However, for (S)-warfarin 7-hydroxylation, some OR-dependence was maintained even when the reaction was supported by cumene hydroperoxi de. Finally, we compared CYP2C9 activity and CYP2C9 protein levels for human lymphoblast expressed (high OR to P450 ratio) to human liver mi crosomes using immunoblotting and enzyme selective substrates. Human l iver microsomal CYP2C9 and human lymphoblast-expressed CYP2C9 showed c omparable amounts of activity per unit enzyme, This final observation indicates that the high OR to P450 ratio is the preferred model and pr edicts that the R144C change in human liver microsomal CYP2C9 should m arkedly reduce the rates of substrate metabolism, The implications of these observations for the interpretation of results with cDNA-express ed enzymes is discussed.