Human cytochrome P450 2C9 (CYP2C9) is important in the metabolism of non-steroidal anti-inflammatory compounds such as diclofenac, the antidiabetic agent tolbutamide and other clinically important drugs, many of which are weakly acidic. Multiple sequence alignment of CYPs identified CYP2C9 Asp293 as corresponding to Asp301 of CYP2D6, which has been suggested to play a role in the binding of basic substrates to the latter enzyme. Replacement of Asp293 with Ala (D293A) decreased activity by more than 90%, and led to an approx. 3- to 10-fold increase in Km values for the three test substrates tolbutamide, dextromethorphan and diclofenac. Conservative replacement of the carboxyl side chain in a Glu (D293E) mutant produced no significant changes in Km values and slight increases in kcat values. Changes in regiospecificity were observed for both the Ala and Glu substitutions; low levels of both dextromethorphan O- and N-demethylation were observed in the D293A mutant, whereas increased preference for O-demethylation was observed for the D293E mutant. Expression of constructs coding for Asn (D293N) and Gln (D293Q) substitutions failed to form a P450 correctly. Our analysis suggests a structural role for the carboxyl side chain of Asp293 in CYP2C9 substrate binding and catalysis. The conservation of an Asp residue in other CYP families in a position equivalent to Asp293 indicates a common mechanism for maintaining the active-site architecture.
Abbreviations used: CYP, cytochrome P450; D293A, Asp293→Ala; D293E, Asp293→Glu; hCPR, human NADPH:CYP oxidoreductase.