The roles of individual amino acids in altering substrate specificity of the P450 2a4/2a5 enzymes

A single amino acid substitution is sufficient to alter substrate specificity of P450 enzymes. Mouse P450 2a5, for example, has its substrate specificity converted from coumarin 7- to testosterone 15 alpha-hydroxylase activity by the substitution of Phe at position 209 to Leu. Furthermore, placing Asn at this position confers a novel corticosterone 15 alpha-hydroxylase activity to this P450. Recent site-directed mutational studies show the presence of the topologically common residues, each of which can determine the specificities of various mammalian P450s. For instance, residue 209 (in 2a5) corresponds to a residue at position 206 in rat P4502B1 that regulates its steroid hydroxylase activity. High substrate specificity often observed in an individual P450, therefore, can be determined and altered by the identities of a few critical residues. The structural flexibility of the substrate-heme pocket may also provide P450 enzymes with the ability to display a broad range of substrate specificities. Understanding the underlying principles whereby the flexible pocket determines P450 activities may lead us to the prediction of P450 activities based on the identities of key amino acid residues.