AFFINITY RADIOLABELING IDENTIFIES PEPTIDES AND AMINO-ACIDS ASSOCIATEDWITH SUBSTRATE-BINDING IN HUMAN PLACENTAL 3-BETA-HYDROXY-DELTA-5-STEROID DEHYDROGENASE

Purified human placental 3beta-hydroxy-DELTA5-steroid dehydrogenase (3 beta-HSD) was affinity radiolabeled by 2alpha-bromo[2'-C-14]acetoxypro gesterone (2alpha-BAP) in the presence or absence of 3beta-HSD substra te, pregnenolone. The substrate steroid substantially protects 3beta-H SD activity from inactivation by 2alpha-BAP. Tryptic peptides of unpro tected and substrate-protected radioalkylated enzyme were purified by high pressure liquid chromatography. The amino acid sequence of each r adiolabeled peptide was determined and localized within the cDNA-deriv ed primary structure of the enzyme. According to the percent total rad ioactivity associated with each of four radiolabeled peaks separated b y high pressure liquid chromatography, two peptides were protected by substrate from affinity radioalkylation by 2alpha-BAP. The first, 251G QFYYISDDTPHQSYDNLNYTLSK274, was produced by tryptic cleavage at Arg-25 0 and Lys-274 (the Arg-250 peptide) and contained radiolabeled His262. The second, 176NGGTLYTCALR186, was produced by tryptic cleavage at Ly s-175 and Arg-186 (the Lys-175 peptide) and contained radiolabeled Cys 183. Based on amino acid analysis to quantitate radioactivity incorpor ated per nmol of peptide, substrate steroid decreased the radiolabelin g of His262 in the Arg-250 peptide by 3.6-fold and decreased the radio labeling of Cys183 in the Lys-175 peptide by 3.7-fold. Three minor rad iolabeled peptides (the NH2-terminal, Arg-71, and Arg-196 tryptic pept ides) were also identified in the primary structure, but pregnenolone did not diminish their affinity radioalkylation. These observations in dicate that the Arg-250 and Lys-175 peptides are involved in substrate binding and suggest that His262 and Cys183 are in close proximity in the three-dimensional structure of the enzyme.