Spatial relationship of steroid and cofactor at the active site of human placental estradiol 17 beta-dehydrogenase.

Two affinity-labeling steroids (2-bromo[2'-14C]acetamidoestrone methyl ether and 16 alpha-bromo[2'-14C]acetoxyestradiol 3-methyl ether) which bear their reagent groups on the A- and D-ring of the molecule, respectively, and which are both substrates, have been used to elucidate spatial relationships of steroid and cofactor as they undergo the reversible binding step at the active site of human placental estradiol 17 beta-dehydrogenase. The 2-derivative alkylates its evolutive cofactor (NADH) in the presence of the enzyme but not in the absence of enzyme. The rate of cofactor alkylation increases with increasing quantities of enzyme and is slowed by estrone which competes for the enzyme-active site. To the contrary, the 16 alpha-derivative does not detectably alkylate its evolutive cofactor (NAD+). The product of cofactor alkylation by 2-bromoacetamidoestrone methyl ether was treated to effect hydrolytic removal of the adenine moiety from the remainder of the cofactor, reduction of the steroid 17-keto group, and crystallization. The final crystalline product has been identified as 2-[2'-6N-adenyl]acetamidoestradiol 3-methyl ether (IUPAC name: N-(3-methoxy-17 beta-hydroxy-1,3,5(10)-estratrien-2-yl)-2-(purin-6-ylamino) acetamide) by ultraviolet, infrared, NMR, and mass spectral analysis.