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A more recent version of this article appeared on November 21, 2001
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M108355200v1
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Papers In Press, published online ahead of print September 18, 2001
J. Biol. Chem, 10.1074/jbc.M108355200
Submitted on August 29, 2001
Revised on September 18, 2001
Accepted on September 18, 2001

Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila

Rebecca D. Miles, Prabha P. Iyer, and James G. Ferry

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

Corresponding Author: jgf3{at}psu.edu

Acetate kinase catalyzes the magnesium-dependent transfer of the g-phosphate of ATP to acetate. The recently determined crystal structure of the M. thermophila enzyme identifies it as a member of the sugar kinase/Hsc70/actin superfamily based on the fold and the presence of five putative nucleotide and metal binding motifs that characterize the superfamily. Residues from four of these motifs in M. thermophila acetate kinase were selected for site-directed replacement and analysis of the variants. Replacement of Asp 148 and Asn 7 resulted in variants with catalytic efficiencies less than 1 % that of the wild-type enzyme indicating that these residues are essential for activity. Glu 384 was also found to be essential for catalysis. A 30-fold increase in the magnesium concentration required for half-maximal activity of the E384A variant relative to that of wild-type implicated Glu 384 in magnesium binding. The kinetic analysis of variants and structural data are consistent with nonessential roles for active site residues Ser 10, Ser 12, and Lys 14 in catalysis. The results are discussed with respect to the acetate kinase catalytic mechanism and the relationship to other sugar kinase/Hsc70/actin superfamily members.


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