INHERENT VERSATILITY OF P-450 OXYGENASE - CONFERRING DEHYDROEPIANDROSTERONE HYDROXYLASE-ACTIVITY TO P-450 2A-4 BY A SINGLE AMINO-ACID MUTATION AT POSITION-117

Mouse steroid 15 alpha hydroxylase P-450 2a-4 is restricted in its sub strate specificity to the Delta(4), 3-ketone steroids such as androste nedione. As a result, the P-450 exhibits little hydroxylase activity t oward Delta(5), 3 hydroxysteroids including dehydroepiandrosterone (DH EA). A single amino acid mutation of Ala at position 117 to Val, howev er, is enough to confer a high DHEA hydroxylase activity to P-450 2a-4 with 7 alpha-OH DHEA as one of the two major hydroxylated metabolites . Mouse coumarin 7-hydroxylase P-450 2a-5 contains Val at position 117 , but it exhibits very low DHEA hydroxylase activity. P-450 2a-5 acqui res high DHEA hydroxylase activity, however, by a mutation of Phe-209 to Asn. Moreover, the mutant P-450 2a-5 loses its activity when Val is replaced by Ala at position 117. The residue at position 117, therefo re, plays the principal role in the determination of the DHEA hydroxyl ase activity of the P-450s. Conversely, mutations at residue 117 have little effect on the androstenedione hydroxylase activities of the P-4 50s. Further modeling of the DHEA binding orientation in the substrate -heme pocket of bacterial P-450cam (Iwasaki, M., Darden, T., Pedersen, L., Davis, D. G., Juvonen, R. O., Sueyoshi, T., and Negishi, M. (1993 ) J. Biol. Chem. 268, 759-762) provides support for the hypothesis tha t the type of residue at position 117 determines the steroid-substrate speci ficity of the P-450 depending on the substituent at the C3 posi tion of steroid molecule.