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Dissociation of Hydroxylase and Lyase Activities by Site-Directed Mutagenesis of the Rat P450₁₇

Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health Bethesda, Maryland 20892

Address requests for reprints to: Dr. Maria L. Dufau, NIH, Building 10, Room B1-L400, Bethesda, Maryland 20892.

Abstract

Site-directed mutagenesis of the arginine-rich region within the putative active site of the rat testicular P450₁₇ (17-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-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 Arg³⁶¹Ala, Arg³⁶¹Lys, 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.

Received for publication April 22, 1991. Revision received June 12, 1991. Accepted for publication July 11, 1991.

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