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Affinity Labeling of Steroid Binding Sites

STUDY OF THE ACTIVE SITE OF 20-HYDROXYSTEROID DEHYDROGENASE WITH 6- AND 11-BROMOACETOXYPROGESTERONE

From the ¹ From the Department of Obstetrics and Gynecology and the Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110

6- and 11-Bromoacetoxyprogesterone were synthesized to further characterize the active site of 20-hydroxysteroid dehydrogenase from Streptomyces hydrogenans. Both of the affinity-labeling steroids react with cysteine, histidine, methionine, and 2-mercaptoethanol in 0.05 m phosphate buffer at pH 7.0, 25°, and are substrates for the enzyme, with apparent K_m values of 2.0 x 10^-5 and 2.78 x 10^-5 m, respectively. 6- and 11-Bromoacetoxyprogesterone inactivate 20-hydroxysteroid dehydrogenase (EC 1.1.1.53) in an irreversible and time-dependent manner. Inactivation follows pseudo-first order kinetics with t_1/2 values of 22 min and 12 hours, respectively. 6-[2-³H]Bromoacetoxyprogesterone and 11-[2-³H]bromoacetoxyprogesterone were synthesized in order to radiolabel the enzyme active site. The amount of radioactivity incorporated during inactivation indicated that each steroid labels a single amino acid. Amino acid analysis of acid hydrolysates of the radiolabeled enzyme revealed that 6-bromoacetoxyprogesterone reacts with a cysteine residue and 11-bromoacetoxyprogesterone reacts with a methionine residue within the active site. The degree of mobility of a steroidal substrate at the active site is discussed on the basis of our earlier and present affinity-labeling experiments.

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