Abstract

The amino acid residues affecting the substrate specificity of human cytochrome P450 CYP2C9 and CYP2C19 for their metabolic activities were investigated using chimeras and mutant enzymes, which were constructed by replacing the corresponding residues. Although CYP2C19 showed nearly the same tolbutamide hydroxylase activity as CYP2C9, the activities for the CYP2C19(H99I) mutant and the chimeras that replaced residues 1–212 were much lower than those for CYP2C19. The activities of the CYP2C19(H99I) mutant and the chimeras that replaced residues 228–340 were lower than those for CYP2C19 toward S-mephenytoin, aminopyrine, and testosterone. These results suggest that residues in substrate recognition site (SRS) 3 and 4 are important for the substrate specificity, whereas His99 is important in the substrate binding of CYP2C19. For the 4′-hydroxylation of diclofenac, CYP2C9 had a lowerK_m and a higherV_max than CYP2C19. Although theV_max values for the CYP2C9(1–288)/CYP2C19(289–490) chimera and the CYP2C9(I99H, V292A, F295L, I331V) mutant were comparable to those for CYP2C9, itsK_m value was comparable to that for CYP2C19. The V_max andK_m values for the CYP2C19(1–288)/CYP2C9(289–490) chimera were comparable to those for CYP2C19, and the activity by CYP2C9(1–230)/CYP2C19(231–490) chimera was negligible. These results suggest that the residues 292, 295, and/or 331 of CYP2C9 are essential for the recognition of substrate in CYP2C9 and that the residues of 231–288 including SRS 3 are important for the metabolizing capacity of CYP2C9.