Human placental estradiol 17 beta-dehydrogenase: sequence of a histidine-bearing peptide in the catalytic region.
The amino acid sequence of an octapeptide from the catalytic site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was established by affinity-labeling techniques. The enzyme was inactivated separately by 12 beta-hydroxy-4-estrene-3,17-dione 12-(bromo[2-14C]acetate) and 3-methoxyestriol 16-(bromo[2-14C]acetate) at pH 6.3. The inactivations, in both cases, followed pseudo-first-order kinetics with half-times for the 12 beta and 16 alpha derivatives being 192 and 68 h, respectively. Both derivatives are known substrates that inactivate in a time-dependent, irreversible manner and that modify cysteine residues to form (carboxymethyl)cysteine and histidine residues to form either N tau- or N pi-(carboxymethyl)histidine. The inactivated enzyme samples were separately reduced, carboxymethylated, and digested with trypsin. The tryptic digests were applied to Sephadex G-50 and the radioactive N tau- and N phi-(carboxymethyl)histidine-bearing peptides identified. The peptides were further purified by cation-exchange chromatography and gel filtration. Final purification was achieved by HPLC prior to sequencing. It was determined that both steroid derivatives modified either of the two histidine residues in the peptide Thr-Asp-Ile-His-Thr-Phe-His-Arg. These histidines are different from a histidine that was previously shown to be alkylated by estrone 3-(bromoacetate) and that was presumed to proximate the A ring of the bound steroid. It is concluded that the two histidine residues identified in the present study proximate the D ring of the steroid as it binds at the active site and may participate in the hydrogen transfer effected by human placental estradiol 17 beta-dehydrogenase.
Murdock
G L
GL
Chin
C C
CC
Warren
J C
JC
D015067:17-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification*; D000345:Affinity Labels / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites; D002250:Carbon Radioisotopes; D004960:Estradiol Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D005260:Female; D006639:Histidine; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding; D011487:Protein Conformation; D014357:Trypsin
D000345:Affinity Labels; D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D006639:Histidine; D015067:17-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases; D014357:Trypsin