Amino acid 305 determines catalytic center accessibility in CYP3A4

Site-directed mutagenesis has been used to replace alanine 305 with phenyla lanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and test osterone metabolism have been determined for both mutants and wildtype CYP3 A4, 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 a nd from 3.2 to 1.2 pmol product/min/pmol P350, respectively. The Vm, for te stosterone 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 ext ent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S50 incre ased from 9 to 42 mu M. The K-i for ketoconazole, a CYP3A4 selective inhibi tor, 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, r espectively, Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 1 5-fold by the A305F mutation. In contrast, the A305S mutation increased tes tosterone 6 beta-hydroxylase (V-max = 2.9 pmol product/min/pmol P450) and e rythromycin N-demethylase (V-max = 5.1 pmol product/min/pmol P450) activiti es, but reduced nifedipine oxidase activity (V-max = 4.6 pmol product/min/p mol P450). K-i values for ketoconazole and other azole inhibitors were unch anged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the ca talytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.