Molecular modelling of members of the P4502A subfamily: application to studies of enzyme specificity.

1. Using the recently published crystal structure of a bacterial P450, namely 102 (also termed P450bm3), as a template molecular models of mammalian 2A1, 2A4, 2A5 and 2A6 were constructed. 2. Substrate interaction studies demonstrated that in keeping with known catalytic activities the putative binding sites of mouse hepatic P4502A4 and 2A5 oriented testosterone for 15 alpha-hydroxylation and coumarin for 7-hydroxylation respectively. 3. Substrate interaction studies with the putative binding site of human liver P4502A6 demonstrated that coumarin was oriented for 7-hydroxylation. However, in keeping with previous site-directed mutagenesis studies with P4502A4 and 2A5, changing a single phenylalanine residue to leucine in 2A6 gave rise to a mutant enzyme, which could bind testosterone as a substrate for 15 alpha-hydroxylation rather than coumarin. 4. Substrate interaction studies with the putative binding site of rat hepatic P4502A1 suggested that this isoenzyme would hydroxylate coumarin at the 3- rather than at the 7-position. 5. The results of these molecular modelling studies demonstrate that apparently minor modifications to P4502A subfamily amino acid sequences can result in major alterations in enzyme specificity. 6. Molecular modelling is thus a useful technique that can aid in elucidating substrate specificities of P450 isoenzymes and species differences in xenobiotic metabolism. The technique can also be utilized to complement site-directed mutagenesis studies in order to identify critical structural features of P450s and other enzymes.