Abstract
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.
Highlights
IntroductionModified steroids have been employed for irreversible inactivation of steroid alcohol dehydrogenases by forming covalently linked compounds with the enzymes ((1) and references cited therein)
From the Laboratory of Human Reproduction and Reproductive Biology and the Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115
Inactivation of Estradiol Dehydrogenase by 3-Bromoacetoxyestrone and Retardation of Inactivation by Substrates and Cofactors-Incubation of estradiol dehydrogenase in the presence of excess 3-bromoacetoxyestrone at pH 6.5 resulted in rapid inactivation that followed pseudo-first order kinetics and led to about 85%) inactivation
Summary
Modified steroids have been employed for irreversible inactivation of steroid alcohol dehydrogenases by forming covalently linked compounds with the enzymes ((1) and references cited therein). In these studies, structural analogy, stoichiometric inactivation, acceptability as substrate, and inhibition of the inactivation by normal substrates or cofactors provided circumstantial evidence that the modified steroid was bound to an amino acid residue at or near the substrate-binding or catalytic site. Direct evidence for binding of an affinity label at the active center of an enzyme would be obtained if, following covalent modification, the enzyme could catalyze its normal chemical reaction using its covalently bound ligand as (co)substrate. Pons et al [3] reported preliminary evidence that 3-iodoacetoxyestrone behaves as an active site-directed, irreversible inhibitor of human placental estradiol-17fi’ dehy-
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