title: Affinity labeling in the presence of the reduced diphosphopyridine nucleotide NADH identifies peptides associated with the activities of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase.
abstract: OBJECTIVE
  We sought to identify peptides associated with activity in the primary structure of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase/isomerase (3 beta-HSD/isomerase).
  
  
  METHODS
  Purified human placental 3 beta-HSD/isomerase was affinity-radioalkylated by 2 alpha-bromo [2'-14C]acetoxyprogesterone (2 alpha-[14C]BAP) in the presence or absence of the reduced diphosphopyridine nucleotide, NADH. NADH protected both 3 beta-HSD and isomerase from inactivation by 2 alpha-[14C]BAP. Tryptic peptides of unprotected and NADH-protected radioalkylated enzyme were purified by high-pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme.
  
  
  RESULTS
  According to the sequence analyses, NADH shifted radioalkylation by 2 alpha-[14C]BAP away from the Arg-250 peptide (251GQFYYISDDTPHQSYDNLNYTLSK274) and toward the Lys-135 tryptic peptide (136EIIQNGHEEEPLENTWPAPYPHSK159). Based on amino acid analysis to quantitate radioactivity incorporated per nmol peptide, NADH decreased the radiolabeling of His262 in the Arg-250 peptide by 8.2-fold. His142 in the Lys-135 peptide was radiolabeled by 2 alpha-[14C]BAP only in the presence of NADH.
  
  
  CONCLUSIONS
  We have previously reported that the substrate pregnenolone blocks the inactivation of 3 beta-HSD by 2 alpha-[14C]BAP through the protection of His262 in the Arg-250 peptide. Protection by NADH against the inactivation of isomerase as well as 3 beta-HSD is evidence that 2 alpha-[14C]BAP binds at the active sites of both enzyme activities. Because the same Arg-250 peptide has been affinity-alkylated in studies that targeted each of the two activities, we propose that the 3 beta-HSD and isomerase reactions are catalyzed in this region of the enzyme protein.
authors.affiliation: Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Thomas
authors.forename: W E
authors.initials: WE
authors.lastname: Nash
authors.forename: M W
authors.initials: MW
authors.lastname: Crankshaw
authors.forename: R C
authors.initials: RC
authors.lastname: Strickler
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D009243:NAD / Q000032:analysis*; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000032:analysis*; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011375:Progesterone Reductase / Q000737:chemistry*; D013251:Steroid Isomerases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014357:Trypsin
chemical_list: C021425:3 beta-hydroxysteroid oxidoreductase-delta(5) 3-ketosteroid isomerase; D000596:Amino Acids; D009097:Multienzyme Complexes; D010455:Peptides; D009243:NAD; D011375:Progesterone Reductase; D014357:Trypsin; D013251:Steroid Isomerases