Arginines 97 and 108 in CYP2C9 are important determinants of the catalyticfunction

Human cytochrome P450 2C9 (CYP2C9) is one of the major drug metabolising en zymes which exhibits a broad substrate specificity. The B-C loop is located in the active-site but has been difficult to model, owing to its diverse a nd flexible structure. To elucidate the function of the B-C loop we used ho mology modelling based on the Cyp102 structure in combination with function al studies of mutants using diclofenac as a model substrate for CYP2C9. The study shows the importance of the conserved arginine in position 97 and th e arginine in position 108 for the catalytic function. The R97A mutant had a 13-fold higher K-m value while the V-max was in the same order as the wil d type. The R108 mutant had a 100-fold lower activity with diclofenac compa red to the wild-type enzyme. The other six mutants (S95A, F100A, L102A, E10 4A, R105A, and N107A) had kinetic parameters similar to the CYP2C9 wild-typ e. Our homology model based on the CYP102 structure as template indicates t hat R97, L102, and R105 are directed into the active site, whereas R108 is not. The change in catalytic function when arginine 97 was replaced with al anine and the orientation of this amino acid in our homology model indicate s its importance for substrate interaction. (C) 2000 Academic Press.