Abstract
Mouse steroid 15 alpha-hydroxylase P-450 2a-4 is restricted in its substrate specificity to the delta 4, 3-ketone steroids such as androstenedione. As a result, the P-450 exhibits little hydroxylase activity toward delta 5, 3-hydroxysteroids including dehydroepiandrosterone (DHEA). A single amino acid mutation of Ala at position 117 to Val, however, 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 acquires 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, therefore, plays the principal role in the determination of the DHEA hydroxylase activity of the P-450s. Conversely, mutations at residue 117 have little effect on the androstenedione hydroxylase activities of the P-450s. 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 that the type of residue at position 117 determines the steroid-substrate specificity of the P-450 depending on the substituent at the C3 position of steroid molecule.
Highlights
From the National Institute of Environmental Health Sciences, National Institutes of Health, Research %angle Park, North Carolina27709
Further modeling tiocofsterone is close to Tyr-96 of P-450cam; the residueknown to the DHEA bindingorientationinthesubstrate-heme form a hydrogen bond with the 2-carbonyl group of the subpocket of bacterial P-450cam
Substrate specificity of mammalian P-450 depending on the type of group at the C3 position of the steroid molecule
Summary
Among the 5 different amino acids within the amino-terminal halvesof the P-450s, residue 117 is requiredfor both P-450 2a-4 and P-450 2a-5 to maintain their respective hydroxylase activities [2].To examine DHEA hydroxylase activityas a function of the residue identitayt position 117(Ala and Val in P-450 in P-450 2a-5 with Ser-102 i n P-450cam. Since camphor is held in the substrate-heme pocket of P-450cam by interaction with multiple hydrophobic amino acids (Phe-87, Tyr-96, Leu-244, Val-247, and Val-295) and by the hydrogen bonding of its carbonyl group to Tyr-96 [18],a steroid molecule in a mammalian P-450 may similarlybe expected to interact with multiple aminoacid residues to maintain its binding orientation in the pocket.
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