Sulfuryl Transfer: The Catalytic Mechanism of Human Estrogen Sulfotransferase*
- From the ‡Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 and the§Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294
Abstract
Estrogen sulfotransferase (EST) catalyzes the transfer of the sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to 17β-estradiol (E2). The sulfation of E2 prevents it from binding to, and thereby activating, the estrogen receptor. The regulation of EST appears to be causally linked to tumorigenesis in the breast and endometrium. In this study, recombinant human EST is characterized, and the catalytic mechanism of the transfer reaction is investigated in ligand binding and initial rate experiments. The native enzyme is a dimer of 35-kDa subunits. The apparent equilibrium constant for transfer to E2 is (4.5 ± 0.2) × 103 at pH 6.3 andT = 25 ± 2 °C. Initial rate studies provide the kinetic constants for the reaction and suggest a sequential mechanism. E2 is a partial substrate inhibitor (K i = 80 ± 5 nm). The binding of two E2 per EST subunit suggests that the partial inhibition occurs through binding at an allosteric site. In addition to providing the dissociation constants for the ligand-enzyme complexes, binding studies demonstrate that each substrate binds independently to the enzyme and that both the E·PAP·E2S andE·PAP·E2 dead-end complexes form. These results strongly suggest a Random Bi Bi mechanism with two dead-end complexes.
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM54469 (to T. S. L.) and GM38953 (to C. N. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Dept. of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2857; Fax: 718-430-8565.
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↵1 The abbreviations used are: PAPS, 3′-phosphoadenosine 5′-phosphosulfate; PAP, adenosine 3′,5′-diphosphate; ER, estrogen receptor; E2, 17β-estradiol; E2S, 17β-estradiol 3-sulfate; EST, estrogen sulfotransferase; DTT, dithiothreitol.
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- Received January 23, 1998.
- Revision received February 13, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
