Synthesis of 4-Bromoacetamidoestrone Methyl Ether and Study of the Steroid Binding Site of Human Placental Estradiol 17/3=Dehydrogenase*

To further characterize the active site of human placental oestradiol17/3-dehydrogenase (EC 1.1.1.62), we have synthesized 4-bromoacetamidoestrone methyl ether. Starting with estrone, synthetic steps involved nitration, isolation of the I-nitro derivative with subsequent methylation, selective reduction of the nitro group, and coupling of the resulting 4-amino group with bromoacetic acid. The affinity-labeling steroid is a substrate for the enzyme with an apparent K, of 1.18 x 10m4 M and apparent V,,,,, value of 0.79 nmol/min/ pg of enzyme. It inactivates the enzyme in a time-dependent, irreversible manner which follows pseudo-first order kinetics. Further, inactivation conducted with varying steroid concentrations displays saturation kinetics (with an apparent Ki of 1.41 x 10m3 M) and indicates that formation of a noncovalent binary complex precedes the alkylation step. When 9.3 x 10m7 M enzyme is inactivated by 1.4 x 10m4 M 4-bromoacetamidoestrone, presence of equimolar concentration of e&radio& or 2.8 x 10e4 M concentrations of NAD+ markedly slow the rate of inactivation. Similar concentrations of bromoacetate are without effect. With the use of 4-bromo[2’-3Hlacetamidoestrone methyl ether, stoichiometry of inactivation indicates that 1.7 mol of carboxymethyl group are incorporated for each mol of enzyme dimer (M, = 68,000) inactivated with or without cofactor. After inactivation with radiolabeled steroid, amino acid analysis of acid hydrolysates reveals carboxymethylcysteine and carboxymethyllysine. When the enzyme is inactivated in the presence of excess NADP+, only the lysyl residue is carboxymethylated. We conclude that the e&radio1 17P-dehydrogenase has lysyl and cysteinyl residues which proximate the lower portion of the steroid A ring as it binds at the active site and that the accessibility of the cysteinyl residue is eliminated (either by direct interposition or conformational change) when the cofactor binding site is occupied by NADP+.