Catalytic competence, a new criterion for affinity labeling. Demonstration of the reversible enzymatic interconversion of estrone and estradiol-17 beta covalently bound to human placental estradiol-17 beta dehydrogenase.
Human placental estradiol-17beta dehydrogenase is rapidly inactivated upon treatment with 3-bromoacetoxyestrone. Pseudo-first order kinetic data are obtained and inactivation is accompanied  by incorporation of 1 mol of 3-acetoxyestrone/mol of subunit (Mr =34,000). Treatment of the inactivated enzyme with (4S)-[4-2H]DPNH results in the formation of covalently bound [17alpha-2H]estradiol-17beta, which can be released by hydrolysis and identified by gas chromatography-mass sepctrometry. When (4R)-[4-2H]DPNH was used, deuterium was not transferred. Thus, the normal stereochemistry of hydridetransfer is preserved for both partners. After treatment with p-mercuribenzoate, affinity-labeled estradiol-17beta dehyrogenase is no longer able to caralyze reduction its covalently bound estrone; in the presence of DPNH and native enzyme, however, reduction occurs, demonstrating that affinity-labeled enzyme can itself serve as subtrate for native estradiol-17beta dehydrogenase. The reversible enzymatic interconversion of covalently bound estrone was demonstrated using a transhydrogenase assay. The ability of an enzyme to catalyze its normal reaction with a covalently bound substrate is termed catalytic competence, and is considered to be a new criterion for affinity labeling.
D000345:Affinity Labels; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D003903:Deuterium; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism; D004960:Estradiol Dehydrogenases / Q000378:metabolism*; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D008626:Mercuribenzoates; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D013237:Stereoisomerism; D014316:Tritium
D000345:Affinity Labels; D002250:Carbon Radioisotopes; D008626:Mercuribenzoates; D009243:NAD; D014316:Tritium; D004970:Estrone; D004958:Estradiol; D009249:NADP; D003903:Deuterium; D006913:Hydroxysteroid Dehydrogenases; D004960:Estradiol Dehydrogenases