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J. Biol. Chem., Vol. 282, Issue 30, 22112-22121, July 27, 2007

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Structural Mechanism for Substrate Inhibition of the Adenosine 5'-Phosphosulfate Kinase Domain of Human 3'-Phosphoadenosine 5'-Phosphosulfate Synthetase 1 and Its Ramifications for Enzyme Regulation^*

Nikolina Sekulic¹, Manfred Konrad², and Arnon Lavie¹³

From the Department of Biochemistry and Molecular Genetics, University of Illinois, Chicago, Illinois 60607 and the Max Planck Institute for Biophysical Chemistry, Göttingen D-37077, Germany

In mammals, the universal sulfuryl group donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is synthesized in two steps by a bifunctional enzyme called PAPS synthetase. The APS kinase domain of PAPS synthetase catalyzes the second step in which APS, the product of the ATP-sulfurylase domain, is phosphorylated on its 3'-hydroxyl group to yield PAPS. The substrate APS acts as a strong uncompetitive inhibitor of the APS kinase reaction. We generated truncated and point mutants of the APS kinase domain that are active but devoid of substrate inhibition. Structural analysis of these mutant enzymes reveals the intrasubunit rearrangements that occur upon substrate binding. We also observe intersubunit rearrangements in this dimeric enzyme that result in asymmetry between the two monomers. Our work elucidates the structural elements required for the ability of the substrate APS to inhibit the reaction at micromolar concentrations. Because the ATP-sulfurylase domain of PAPS synthetase influences these elements in the APS kinase domain, we propose that this could be a communication mechanism between the two domains of the bifunctional enzyme.

Received for publication, February 27, 2007 , and in revised form, May 8, 2007.

The atomic coordinates and structure factors (code 2pey and 2pez) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract W-31-109-Eng-38. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4.

¹ Supported by National Institutes of Health Grant AI046943.

² Supported by the Max-Planck Society.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, 900 South Ashland Ave., MBRB Room 1108, University of Illinois, Chicago, IL 60607. Tel.: 312-355-5029; Fax: 312-355-4535; E-mail: lavie{at}uic.edu.

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