Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO- + ATP⇆CH3CO2PO32- + ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from Escherichia coil indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273-280, 19741. Alignment of the amino acid sequences for the acetate kinases from E. coli (Bacteria domain), Methanosarcina thermophila (Archaea domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in the M. thermophila enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced in E. coli and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the K(m) values for acetate and ATP with only moderate decreases in k(cat), which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by N- ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to N-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased the K(m) value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of bacteriology|
|Publication status||Published - Mar 1 1998|
All Science Journal Classification (ASJC) codes
- Molecular Biology