Amino acid 305 determines catalytic center accessibility in CYP3A4

Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wildtype CYP3A4, The A305F mutation abolished diazepam oxidase activity and reduced the S-50 and V-max for erythromycin N-demethylase activity from 17 to 10 mu M and from 3.2 to 1.2 pmol product/min/pmol P350, respectively. The Vm, for testosterone 6 beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P350, whereas the S-50 increased from 33 to 125 mu M. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S50 increased from 9 to 42 mu M. The K-i for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 mu M, from 0.052 to 0.73 mu M, and from 0.043 to 2.2 mu M by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively, Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6 beta-hydroxylase (V-max = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V-max = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V-max = 4.6 pmol product/min/pmol P450). K-i values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.