Dissociation of hydroxylase and lyase activities by site-directed mutagenesis of the rat P45017 alpha.
Site-directed mutagenesis of the arginine-rich region within the putative active site of the rat testicular P45017 alpha (17 alpha-hydroxylase cytochrome P-450, the product of P450XVII gene) was performed to identify specific amino acids that contribute to either the hydroxylase or lyase activities catalyzed by this enzyme. The conversion of Arg346 to alanine differentially abolished lyase activity without affecting hydroxylase activity, resulting in an accumulation of the 17 alpha-hydroxylated intermediate, and partial lyase activity was recovered by conversion of this mutant to Lys346. Similar results were obtained with the conversion of Arg357 to alanine, although this mutant also diminished hydroxylase activity, and full lyase and hydroxylase activities were recovered with a lysine in this position. Major reductions in hydroxylase activity were apparent with the conversion of Arg363 to Ala, and this inhibition was reversed by a lysine at position 363. In contrast, differential effects were not observed with the mutants Arg361 Ala, Arg361 Lys, or Tyr334Phe. Both mutations at the Arg361 position resulted in a total loss of hydroxylase and lyase activities, and mutation at the Tyr334 position had no effect on either activity. The identification of specific amino acids that are essential for either the hydroxylase or lyase reaction indicates that the steroid substrate-protein interaction changes during the course of the two consecutive reactions and reveals the potential for separation of the two activities by chemical and biological modulators within the active site region of the P45017 alpha.
Kitamura
M
M
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Buczko
E
E
Dufau
M L
ML
D000446:Aldehyde-Lyases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology; D014162:Transfection
D011994:Recombinant Proteins; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D013254:Steroid 17-alpha-Hydroxylase; D000446:Aldehyde-Lyases