Reactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase affinity alkylated by estrone 3-(bromoacetate): topographic studies with 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether).
Estradiol 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, oxidoreductase activities copurified from the cytosol of human-term placenta as a homogeneous protein (native enzyme), were reactivated at equal rates to 100% activity following complete inactivation in the presence of cofactor (NADPH) with the affinity alkylator estrone 3-(bromoacetate). Reactivation was accomplished by base-catalyzed hydrolysis of steroidal ester-amino acid linkages in the enzyme active site. The rate of enzyme reactivation was pH dependent. In identical studies without NADPH, only 12% of the original enzyme activity was restored. Completely reactivated enzyme was repurified by dialysis. Enzyme in control mixtures (control enzyme) that contained estrone in place of alkylator was treated the same as the reactivated enzyme. Reactivated enzyme exhibited a 6.0-fold lower affinity for common substrates, a 1.8-fold lesser affinity for NAD+ and NADH, and the same affinity for NADP+ and NADPH compared to control enzyme. In incubations that included NADPH, the reactivated enzyme maintained full activity during a 20-h second exposure to estrone 3-(bromoacetate), but in identical incubations without NADPH, the reactivated enzyme was rapidly inactivated at the same rate as the control and native enzymes. The control and reactivated enzymes were inactivated at equal rates by 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether) in the presence or absence of cofactor (NADP+) and exhibited similar Kitz and Wilson inhibition constants for this affinity alkylator. Estrone 3-(bromo[2'-14C]acetate) incubated with native enzyme and NADPH produced radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine.(ABSTRACT TRUNCATED AT 250 WORDS)
D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D015089:20-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D000345:Affinity Labels / Q000494:pharmacology*; D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D004958:Estradiol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D004960:Estradiol Dehydrogenases / Q000378:metabolism*; D004970:Estrone / Q000031:analogs & derivatives*; D005260:Female; D006801:Humans; D006894:Hydroxyestrones / Q000494:pharmacology*; D007202:Indicators and Reagents; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding
D000345:Affinity Labels; D000596:Amino Acids; D006894:Hydroxyestrones; D007202:Indicators and Reagents; D004970:Estrone; D004958:Estradiol; C010870:16 alpha-bromoacetoxyestradiol-3-methyl ether; C034312:estrone 3-bromoacetate; D015067:17-Hydroxysteroid Dehydrogenases; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D004960:Estradiol Dehydrogenases