The affinity alkylators, 11α-bromoacetoxyprogesterone and estrone 3-bromoacetate, modify a common histidyl residue in the active site of human placental 17β,20α-hydroxysteroid dehydrogenase

Abstract

Purified human placental 17β,20α-hydroxysteroid dehydrogenase (native enzyme) was completely inactivated by the affinity alkylator, estrone 3-bromoacetate, in the presence ofcofactor (NADPH). The inactivated enzyme was reactivated to 100% activity by base-catalyzed hydrolysis of the steroidal ester-enzyme conjugate and then repurified by dialysis. Control enzyme in mixtures which contained estrone in place of alkylator was treated the same as the reactivated enzyme. 11α-Bromo[2'- 14C]acetoxy-progesterone, an active site-directed affinity alkylator of the enzyme, produced 5.0-fold less radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine plus 1.4-fold more 1,3- s (carboxymethyl)-histidine in the reactivated enzyme than in the control enzyme. The lesser amount of S-(carboxymethyl)cysteine and greater amount of 1,3- bis(carboxymethyl)histidine resulted from nonspecific interactions between the reactivated enzyme and the progestin radioalkylator. The nonradiolabeled 3-(carboxymethyl)histidine originally produced by estrone 3-bromoacetate in the enzyme active site hindered radioalkylation of this amino acid by 11α-bromop[2'- 14C]acetoxyprogesterone to yield 5-fold less radiolabeled 3-(carboxymethyl)histidine in the reactivated enzyme relative to control enzyme. Thus, the estrogen and progestin affinity alkylators modified a common histidyl residue in the active site. These studies are direct evidence that the estradiol 17β-dehydrogenase and 20α-hydroxysteroid dehydrogenase activities reside at a common locus on a single protein.