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J. Biol. Chem., Vol. 280, Issue 11, 10731-10742, March 18, 2005

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Structural and Kinetic Analyses of Arginine Residues in the Active Site of the Acetate Kinase from Methanosarcina thermophila^*

Andrea Gorrell, Sarah H. Lawrence, and James G. Ferry

From the Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802

Acetate kinase catalyzes transfer of the -phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680–686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5'-[-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al³⁺, and F^- contained a linear array of ADP-AlF₃-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF₃ mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His¹⁸⁰ was in close proximity to AlF₃ is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg²⁴¹ was also found adjacent to AlF₃, consistent with a role for stabilization of the transition state. Kinetic analyses of Arg²⁴¹ and Arg⁹¹ replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.

Received for publication, October 26, 2004 , and in revised form, December 10, 2004.

The atomic coordinates and structure factors (codes 1TUU and 1TUY) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant GM44661, Department of Energy Grant DE-FG02-95ER20198, and the Eberly College of Science at Pennsylvania State University. 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.

Present address: Dept. of Chemistry, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada.

To whom correspondence should be addressed. Tel.: 814-863-5721; Fax: 814-863-6217; E-mail: jgf3{at}psu.edu.

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