Acetate kinase, a member of the acetate and sugar kinase/Hsc 70/actin (ASKHA) structural superfamily, catalyzes the reversible transfer of the gamma-phosphoryl group from ATP to acetate, yielding ADP and acetyl phosphate. A catalytic mechanism for the enzyme from Methanosarcina thermophila has been proposed on the basis of the crystal structure and kinetic analyses of amino acid replacement variants. The Gln43Trp variant was generated to further investigate the catalytic mechanism via changes in fluorescence. The dissociation constants for ADP.Mg2+ and ATP.Mg2+ ligands were determined for the Gln43Trp variant and double variants generated by replacing Arg241 and Arg91 with Ala and Lys. The dissociation constants and kinetic analyses indicated roles for the arginines in transition state stabilization for catalysis but not in nucleotide binding. The results also provide the first experimental evidence for domain motion and evidence that catalysis does not occur as two independent active sites of the homodimer but the active site activities are coordinated in a half-the-sites manner.