The affinity alkylators, 11 alpha-bromoacetoxyprogesterone and estrone 3-bromoacetate, modify a common histidyl residue in the active site of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase.
Purified human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (native enzyme) was completely inactivated by the affinity alkylator, estrone 3-bromoacetate, in the presence of cofactor (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. 11 alpha-Bromo[2'-14C]acetoxyprogesterone, 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-bis(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 11 alpha-bromo[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 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities reside at a common locus on a single protein.
Thomas
J L
JL
Asibey-Berko
E
E
Strickler
R C
RC
D000478:Alkylation; D001665:Binding Sites; D004960:Estradiol Dehydrogenases / Q000037:antagonists & inhibitors; D004970:Estrone / Q000031:analogs & derivatives*; D005260:Female; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006894:Hydroxyestrones / Q000494:pharmacology*; D006908:Hydroxyprogesterones / Q000494:pharmacology*; D006913:Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy
D006894:Hydroxyestrones; D006908:Hydroxyprogesterones; D004970:Estrone; C005669:11 alpha-bromoacetoxyprogesterone; D006639:Histidine; C034312:estrone 3-bromoacetate; D006913:Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases