Affinity Labeling of Steroid Binding Sites

To further study the steroid binding site of 20&hydroxysteroid dehydrogenase (EC 1.1.1.53) by affinity labeling, 21[2’JH]bromoacetylaminoprogesterone was synthesized. The steroid inactivates the enzyme in a time-dependent and irreversible manner which follows pseudo-first order kinetics. Incorporation of 2 mol of 21-[2’-3Hlbromoacetylaminoprogesterone accompanies inactivation of 1 mol of enzyme. Amino acid analysis of the 6 N HCl hydrolysate of the radiolabeled enzyme identified dicarboxymethylhistidine as the main alkylation product. The stability of the amide bond in 21-bromoacetylaminoprogesterone excludes the possibility that dialkylation of the active site histidine residue occurs by a transacylation mechanism. When 2Ophydroxysteroid dehydrogenase is inactivated with 16a-[2’3Hlbromoacetoxyprogesterone in the presence of excess [214Cliodoacetic acid, amino acid analysis of the acid hydrolysate of the radiolabeled enzyme shows that 3H and 14C are present in dicarboxymethylhistidine in a ratio of 1.03:1. A two-step mechanism involving a specific, site-directed, steroid-mediated alkylation followed by a rapid nonspecific alkylation step is proposed to account for dialkylation of an active site histidine residue resulting from affinity labeling of 20/Shydroxysteroid dehydrogenase by 16a-bromoacetoxyprogesterone and 21-bromoacetylaminoprogesterone. 21-Bromoacetylaminoprogesterone, 21-acetylaminoprogesterone, and 16a-carboxyamidoprogesterone are not reduced in the presence of NADH when tested as substrates for the enzyme. Kinetic studies show that all three progesterone amide analogs are competitive inhibitors of cortisone reduction. However, 21-azidoprogesterone which is electronically and sterically similar to 21-acetylaminoprogesterone is a substrate with a V,,, value of 10.5 nmol min-’ pg-’ and an apparent K, value of 7.1 X 10m5 M. 16aZyanoprogesterone, which is structurally analogous to 16a+arboxyamidoprogesterone, has a V,,, value of 9.25 nmol min-’ pg-’ and an apparent K, value of 6.7 x 10e4 M. A mechanism involving unfavorable hydrogen bonding interactions between the