Human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase. Studies with 6 beta-bromoacetoxyprogesterone.
Two soluble enzyme activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, copurified from the cytosol fraction of human term placenta, were identically inactivated by 6 beta-bromoacetoxyprogesterone. This affinity alkylating steroid binds at the enzyme-active site (Km = 866 microM; Vmax = 0.073 mumol/min/mg). Enzyme inactivation by four concentrations of 6 beta-bromoacetoxyprogesterone (molar ratio of steroid to enzyme, 71/1 to 287/1) causes irreversible and time-dependent loss of both the 17 beta- and 20 alpha-activities according to first order kinetics and affirms that the alkylating steroid is an active site-directed inhibitor (KI = 2.7 X 10(-3) M; k3 = 1.6 X 10(-3) s-1). Affinity radioalkylation studies using 6 beta-[2'-14C]bromoacetoxyprogesterone indicate that 2 mol of steroid are bound to each mole of inactivated enzyme dimer (Mr = 68,000). Amino acid analyses of the acid hydrolysate of radioalkylated enzyme show that 6 beta-bromoacetoxyprogesterone carboxymethylates cysteine (56%), histidine (22%), and lysine (8%) residues in the active site. These results are identical with those reported for 2-bromo[2'-14C]acetamidoestrone methyl ether radioalkylation of purified "17 beta-estradiol dehydrogenase." The parallel inactivation of 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase by 6 beta-bromoacetoxyprogesterone further shows that both activities reside at a single enzyme-active site. The radioalkylation profile supports our proposed model of one enzyme-active site wherein the bound progestin and estrogen substrates are inverted, one relative to the other.
D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D015089:20-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D000345:Affinity Labels / Q000494:pharmacology*; D001665:Binding Sites; D004960:Estradiol Dehydrogenases / Q000378:metabolism*; D005260:Female; D006801:Humans; D006908:Hydroxyprogesterones / Q000494:pharmacology*; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding
C005670:6-bromoacetoxyprogesterone; D000345:Affinity Labels; D006908:Hydroxyprogesterones; D015067:17-Hydroxysteroid Dehydrogenases; D015089:20-Hydroxysteroid Dehydrogenases; D043607:20-alpha-Hydroxysteroid Dehydrogenase; D004960:Estradiol Dehydrogenases