RECIPROCAL SIZE - EFFECT RELATIONSHIP OF THE KEY RESIDUES IN DETERMINING REGIOSPECIFICITIES AND STEREOSPECIFICITIES OF DHEA HYDROXYLASE-ACTIVITY IN P450 2A5

Collectively, the P450 2a4/2a5 system hyrdoxylates DHEA in at least th ree positions (7 alpha, 7 beta, and 2 alpha). An individual P450, howe ver, exhibits high specificity to one of these products. Using site-di rected mutagenesis of mP450 2a5 from the wild mouse Mus minutoides and bacterial expression, we have associated the function of residues 117 , 209, and 481 with the respective specificity observed in each P450. Ala at position 117 determines the 7 beta-hydroxylase activity, wherea s Val at this position defines the 2 alpha-hydroxylase activity. Leu a t position 209 is essential for high DHEA 7 alpha-hydroxylase activity . The substitutions of residue 481 with various hydrophobic amino acid s elicited a profound alteration of the specific hydroxylation rates, but did not influence the regio- and stereospecificities at either of the three positions of DHEA. The alterations caused by residue 481 als o depended on the residue identity at position 117 or 209. The results indicate that the sizes of several key residues obey a concerted reci procal relationship whereby the substrate pocket of the P450s adjusts to accommodate DHEA. A limited molecular modeling study successfully c orrelates DHEA binding to experimental DHEA hydroxylase activities for a series of mutants at key positions.