Human placental estradiol 17 beta-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site.
Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity labeled with 17 alpha-estradiol 17-(bromo[2-14C]acetate) (10 microM) or 17 beta-estradiol 17-(bromo[2-14C]acetate) (10 microM). The steroid bromoacetates competitively inhibit the enzyme (against 17 beta-estradiol) with Ki values of 90 microM (17 alpha bromoacetate) and 134 microM (17 beta bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with a t1/2 = 110 min (17 alpha bromoacetate) and t1/2 = 220 min (17 beta bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N pi-(carboxy[14C]methyl)histidine was the modified amino acid labeled in each case. Digestion with trypsin produced peptides that were isolated by reverse-phase high-performance liquid chromatography and found to contain N pi-(carboxy[14C]methyl)histidine. Both the 17 alpha bromoacetate and also the 17 beta bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(pi-CM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involved in catalytic hydrogen transfer at the D-ring of estradiol. Therefore, this histidine was presumed to proximate the A-ring of the bound steroid substrate. The present results suggest that the 17 alpha bromoacetate and 17 beta bromoacetate D-ring analogues of estradiol react with the same active site histidine residue as estrone 3-(bromoacetate), the A-ring analogue of estrone.(ABSTRACT TRUNCATED AT 250 WORDS)
Murdock
G L
GL
Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, Missouri 63110.
Warren
J C
JC
Sweet
F
F
D015067:17-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors*; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004958:Estradiol / Q000378:metabolism*; D004960:Estradiol Dehydrogenases / Q000037:antagonists & inhibitors*; D005260:Female; D006801:Humans; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013379:Substrate Specificity
D000596:Amino Acids; D004958:Estradiol; D015067:17-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases