title: Human placental 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase. Two activities at a single enzyme active site.
abstract: Two soluble enzyme activities, 17 beta-estradiol dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, present in the cytosol fraction of term human placenta, were co-purified with a constant ratio of specific activities, approximating 100:1, respectively. The "pure enzyme" is a single band on sodium dodecyl sulfate disc gel electrophoresis. To evaluate whether catalysis of the estrogen and progestin substrates occurs at a single active site, alkylation studies using 16 alpha-bromoacetoxyprogesterone were designed. This affinity alkylating steroid binds at the enzyme-active site (km 256 microM; Vmax = 0.012 mumol/min/mg), inactivates the enzyme in an irreversible and time-dependent manner which follows pseudo-first order kinetics, and causes coincident loss of both the 17 beta- and 20 alpha-activities. Affinity radioalkylation studies using 16 alpha-[2'-3H]bromoacetoxyprogesterone indicate that 2 mol of steroid bind per mol of inactivated enzyme dimer (Mr = 68,000). Amino acid analyses of the acid hydrolysate of radioalkylated enzyme show that 16 alpha-bromoacetoxyprogesterone dicarboxymethylates a histidyl residue in the active site. These results are identical with those reported for 16 alpha-[2'-3H]bromoacetoxyestradiol 3-methyl ether inactivation and radioalkylation of identically purified "17 beta-estradiol dehydrogenase." Computer graphics were used to construct a model in which: 1) binding of estrogen and progestin substrates at one active site permits stereospecific catalysis; 2) the estrogen and progestin analogs' alkylating side arms have access to a common histidine residue. These observations clearly demonstrate that the catalysis of estrogen and progestin substrates can occur at a single active site of one enzyme.
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D015089:20-Hydroxysteroid Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D004960:Estradiol Dehydrogenases / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D013379:Substrate Specificity
chemical_list: D015067:17-Hydroxysteroid Dehydrogenases; D015089:20-Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases