Vol. 30, Issue 4, 452-456, April 2002

CYP3A4 Active Site Volume Modification by Mutagenesis of Leucine 211

Stephen M. Fowler, John M. Taylor, Thomas Friedberg, C. Roland Wolf, and Robert J. Riley

Physical and Metabolic Science, AstraZeneca R&D Charnwood, Loughborough, United Kingdom (S.M.F., J.M.T., R.J.R.); and Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, United Kingdom (T.F., C.R.W.)

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 6-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.