Abstract
Acetate kinase catalyzes the magnesium-dependent transfer of the gamma-phosphate of ATP to acetate. The recently determined crystal structure of the Methanosarcina 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% of 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 the wild type implicated Glu(384) in magnesium binding. The kinetic analysis of variants and structural data is 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.
Highlights
Acetate kinase, which catalyzes the magnesium-dependent transfer of the ATP ␥-phosphate to acetate (Eq 2), is a central enzyme in the energy-yielding metabolism of anaerobes
The recently determined crystal structure of the Methanosarcina 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
The triple displacement mechanism was challenged when it was shown that phosphorylated acetate kinase from E. coli is a phosphoryl donor to enzyme I of the bacterial phosphotransferase system (6), which suggested an alternate function for phosphorylated acetate kinase in the transport of sugars
Summary
Protein Sequence Analyses—Data bases were searched at the National Center for Biotechnology Information using the BLAST network server (19). Enzyme Activity Assays—The hydroxamate assay, an adaptation of the method of Lipmann and Tuttle (24) and Rose et al (2), indirectly detects the formation of acetyl phosphate from acetate and ATP. This previously described assay (25) was modified such that the final concentrations of the assay components were as follows (unless otherwise noted): 145 mM Tris (pH 7.4), 500 mM potassium acetate, 10 mM MgCl2, 10 mM ATP, and 705 mM hydroxylamine hydrochloride. In the determination of the Km for acetate, the concentration of ATP was 10 mM unless the Km for ATP was determined to be higher than that for the wild-type enzyme, in which case the concentration was increased to 20 mM. Protein samples (1.0 ml) were loaded onto the column that had been pre-equilibrated with 145 mM Tris-HCl buffer, pH 7.0, containing 150 mM NaCl, and the column was developed with a flow rate of 0.5 ml/min
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