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J. Biol. Chem., Vol. 279, Issue 18, 18799-18805, April 30, 2004

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Active Site Mutations and Substrate Inhibition in Human Sulfotransferase 1A1 and 1A3^*

Amanda C. Barnett, Sergey Tsvetanov, Niranjali Gamage, Jennifer L. Martin¶, Ronald G. Duggleby||, and Michael E. McManus**

From the School of Biomedical Sciences, Institute for Molecular Bioscience, and ^||School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia

Human SULT1A1 is primarily responsible for sulfonation of xenobiotics, including the activation of promutagens, and it has been implicated in several forms of cancer. Human SULT1A3 has been shown to be the major sulfotransferase that sulfonates dopamine. These two enzymes shares 93% amino acid sequence identity and have distinct but overlapping substrate preferences. The resolution of the crystal structures of these two enzymes has enabled us to elucidate the mechanisms controlling their substrate preferences and inhibition. The presence of two p-nitrophenol (pNP) molecules in the crystal structure of SULT1A1 was postulated to explain cooperativity at low and inhibition at high substrate concentrations, respectively. In SULT1A1, substrate inhibition occurs with pNP as the substrate but not with dopamine. For SULT1A3, substrate inhibition is found for dopamine but not with pNP. We investigated how substrate inhibition occurs in these two enzymes using molecular modeling, site-directed mutagenesis, and kinetic analysis. The results show that residue Phe-247 of SULT1A1, which interacts with both p-nitrophenol molecules in the active site, is important for substrate inhibition. Mutation of phenylalanine to leucine at this position in SULT1A1 results in substrate inhibition by dopamine. We also propose, based on modeling and kinetic studies, that substrate inhibition by dopamine in SULT1A3 is caused by binding of two dopamine molecules in the active site.

Received for publication, November 10, 2003 , and in revised form, January 28, 2004.

^* This work was supported by National Health and Medical Research Council of Australia Grant 252734. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a fellowship from the Australian Research Council.

^** To whom correspondence should be addressed: Faculty of Biological and Chemical Sciences, Computer Science Building (Bldg. 69, Rm. 304), University of Queensland, Queensland 4072, Australia. Tel.: 61-7-3365-1609; Fax: 61-7-3365-1613; E-mail: m.mcmanus{at}uq.edu.au.

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