CYP3A4 active site volume modification by mutagenesis of leucine 211

The leucine 211 → phenylalanine (L211F) and leucine 211 → tyrosine (L211Y) mutant forms of cytochrome P450 3A4 have been generated by site-directed mutagenesis and expressed functionally in Escherichia coli. Substrate binding affinities (S₅₀ values) for testosterone and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were similar for the mutants and wild-type CYP3A4 (49 and 21 μM for L211F, 35 and 20 μM for L211Y, and 33 and 20 μM for the wild type, respectively). For erythromycin, however, the K_m values determined for the L211F and L211Y mutants were 2.4- and 10.5-fold higher than for the wild type. Furthermore, IC₅₀ values for the inhibition of testosterone 6β-hydroxylation by erythromycin and troleandomycin for L211F were 2.4- and 3.7-fold higher, and those for L211Y were 3.4- and 9.2-fold higher than those measured for the wild type. Conversely, small inhibitors, such as diazepam, exhibited no significant difference in IC₅₀ values between the wild type and the L211F and L211Y mutants. It is proposed that large substrates bound in the catalytic center of CYP3A4 with molecular volumes greater than ∼600 ų were less well accommodated in the altered active sites, resulting in lower association energies and increased IC₅₀ values.