Purification and characterization of homo- and hetero-dimeric acetate kinases from the sulfate-reducing bacterium Desulfovibrio vulgaris.
Two distinct forms of acetate kinase were purified to homogeneity from a sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F. The enzymes were separated from the soluble fraction of the cells on anion exchange columns. One acetate kinase (AK-I) was a homodimer (alpha(S)(2)) and the other (AK-II) was a heterodimer (alpha(S)alpha(L)). On SDS-PAGE, alpha(L) and alpha(S) subunits migrated as bands of 49.3 and 47.8 kDa, respectively, but they had an identical N-terminal amino acid sequence. A rapid HPLC method was developed to directly measure ADP and ATP in assay mixtures. Initial velocity data for AK-I and AK-II were collected by this method and analyzed based on a random sequential mechanism, assuming rapid equilibrium for the substrate binding steps. All kinetic parameters for both the forward acetyl phosphate formation and the reverse ATP formation catalyzed by AK-I and AK-II were successfully determined. The two enzymes showed similar kinetic properties in Mg(2+) requirement, pH-dependence and magnitude of kinetic parameters. These results suggest that two forms of acetate kinase are produced to finely regulate the enzyme function by post-translational modifications of a primary gene product in Desulfovibrio vulgaris.
Department of Biochemistry, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan.
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Yu
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Ishida
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Ozawa
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Akutsu
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Horiike
D000084:Acetate Kinase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D016969:Desulfovibrio vulgaris / Q000201:enzymology* / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D013431:Sulfates / Q000378:metabolism
D021122:Protein Subunits; D013431:Sulfates; D019344:Lactic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000084:Acetate Kinase; D008274:Magnesium