Human placental estradiol 17β-dehydrogenase: evidence for inverted substrate orientation (wrong-way binding) at the active site

Human placental estradiol 17β-dehydrogenase was affinity labeled with 17λ-estradiol 17-(bromo[2-14C]acetate) (10 μM) or 17β-estradiol 17-(bromo[2-14C]acetate) (10 μM). The steroid bromoacetates competitively inhibit the enzyme (against 17β-estradiol) with K/sub i/ values of 90 μM (17α bromoacetate) and 134 μM(17β bromoacetate). Inactivation of the enzyme followed pseudo-first-order kinetics with t/sub 1/2/ = 110 min (17α bromoacetate) and t/sub 1/2/ = 220 min (17β bromoacetate). Amino acid analysis of the affinity radioalkylated enzyme samples from the two bromoacetates revealed that N/sup π/-(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/sup π/-(carboxy[14C]methyl)histidine. Both the 17α bromoacetate and also the 17β bromoacetate modified the same histidine in the peptide Phe-Tyr-Gln-Tyr-Leu-Ala-His(πCM)-Ser-Lys. Previously, the same histidine had been exclusively labeled by estrone 3-(bromoacetate) and shown not to be directly involve 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 17α bromoacetate and 17β bromoacetate D-ring analogue 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 17β-estradiol as it undergoes catalytic hydrogen transfer at the same active site