Human placental estradiol 17.beta.-dehydrogenase: evidence for inverted substrate orientation ("wrong-way" binding) at the active site
- 1 June 1988
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 27 (12) , 4452-4458
- https://doi.org/10.1021/bi00412a036
Abstract
Human placental estradiol 17.beta.-dehydrogenase (EC 1.1.1.62) was affinity labeled with 17.alpha.-estradiol 17-(bromo[2-14C]acetate) (10 .mu.M) or 17.beta.-estradiol 17-(bromo[2-14C]acetate) (10 .mu.M). The steroid bromoacetates competitively inhibit the enzyme (against 17.beta.-estradiol) with Ki values of 90 .mu.M (17.alpha. bromoacetate) and 134 .mu.M (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. Moreover, as each of the estradiol 17-(bromoacetates) undergoes the reversible binding step at the enzyme active site, its D-ring is in a reversed binding position relative to that of the natural substrate 17.beta.-estradiol as it undergoes catalytic hydrogen transfer at the same active site.Keywords
This publication has 15 references indexed in Scilit:
- Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.Proceedings of the National Academy of Sciences, 1982
- Dual activity at an enzyme active site: 3.beta.,20.alpha.-hydroxysteroid oxidoreductase from fetal bloodBiochemistry, 1982
- Identification of two histidyl residues in the active site of human placental estradiol 17.beta.-dehydrogenaseBiochemistry, 1982
- Isolation of histidyl peptides of the steroid-binding site of human placental estradiol 17β-dehydrogenaseSteroids, 1982
- Estradiol 17β-dehydrogenase and 20α-hydroxysteroid dehydrogenase from human placental cytosol: one enzyme with two activities?Steroids, 1980
- Irreversible active-site-directed inhibition of Δ5-3-ketosteroid isomerase by steroidal 17-β-oxiranes. Evidence for two modes of binding in steroid-enzyme complexesBiochemical and Biophysical Research Communications, 1980
- Bifunctional enzyme activity at the same active site: study of 3.alpha. and 20.beta. activity by affinity alkylation of 3.alpha.,20.beta.-hydroxysteroid dehydrogenase with 17-(bromoacetoxy)steroidsBiochemistry, 1980
- Crystallization of human placental estradiol 17beta-dehydrogenase. A new method for crystallizing labile enzymes.Journal of Biological Chemistry, 1976
- Alkylation and Identification of the Histidine Residues at the Active Site of RibonucleaseJournal of Biological Chemistry, 1963
- HUMAN PLACENTAL ESTRADIOL-17-BETA DEHYDROGENASE .1. CONCENTRATION, CHARACTERIZATION AND ASSAY1958