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

OBJECTIVE: We sought to identify peptides associated with activity in the primary structure of human placental 3 beta-hydroxy-Delta(5)-stero id dehydrogenase/isomerase (3 beta-HSD/isomerase). METHODS: Purified h uman placental 3 beta-HSD/isomerase was affinity-radioalkylated by 2 a lpha-bromo[2'-C-14]acetoxyprogesterone (2 alpha-[C-14]BAP) in the pres ence or absence of the reduced diphosphopyridine nucleotide, NADH. NAD H protected both 3 beta-HSD and isomerase from inactivation by 2 alpha -[C-14]BAP. Tryptic peptides of unprotected and NADH-protected radioal kylated enzyme were purified by high-pressure liquid chromatography. T he amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. RE SULTS: According to the sequence analyses, NADH shifted radioalkylatio n by 2 alpha[C-14]BAP away from the Arg-250 peptide ((251)GQFYYISDDTPH QSYDNLNYTLSK(274)) and toward the Lys-135 tryptic peptide ((136)EIIQNG HEEEPLENTWPAPYPHSK(159)). Based on amino acid analysis to quantitate r adioactivity incorporated per nmol peptide, NADH decreased the radiola beling of His(262) in the Arg-250 peptide by 8.2-fold. His(142) in the Lys-135 peptide was radiolabeled by 2 alpha-[C-14]BAP only in the pre sence of NADH. CONCLUSIONS: We have previously reported that the subst rate pregnenolone blocks the inactivation of 3 beta-HSD by 2 alpha[C-1 4]BAP through protection of His(262) in the Arg-250 peptide. Protectio n by NADH against the inactivation of isomerase as well as 3 beta-HSD is evidence that 2 alpha-[C-14]BAP binds at the active sites of both e nzyme activities. Because the same Arg-250 peptide has been affinity-a lkylated in studies that targeted each of the two activities, we propo se that the 3 beta-HSD and isomerase reactions are catalyzed in this r egion of the enzyme protein.