Purification and properties of acetate kinase from Acholeplasma laidlawii.
Acetate kinase (EC 2.7.2.1) was purified from Acholeplasma laidlawii cytoplasm by a combination of ammonium sulfate fractionation, gel filtration, diethylaminoethyl-cellulose chromatography, and affinity chromatography on 8-(6-aminohexylamino)-adenosine 5'-triphosphate conjugated to Sepharose 4B. The enzyme was composed of polypeptide chains of about 50,000 molecular weight as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturating conditions, apparent molecular weights between 64,000 and 130,000 were obtained, depending upon mainly the ionic strength of the test solution. The enzyme had a narrow specificity for phosphate acceptor acids, whereas both purine and pyrimidine nucleoside triphosphates were suitable phosphate donors. Na(+) and K(+) inhibited both acetyl phosphate and adenosine 5'-triphosphate synthesis, and the latter was also inhibited by high concentrations of adenosine 5'-diphosphate and acetyl phosphate. This substrate inhibition was partially abolished by 0.5 M NaCl. The enzyme catalyzed the independent adenosine 5'-diphosphate<-->adenosine 5'-triphosphate and acetate<-->acetyl phosphate exchanges. The rate of the latter was enhanced by the addition of cosubstrate Mg(2+)-adenosine 5'-triphosphate. The high affinity for substrates, except for acetate, indicated that under physiological conditions the direction of the enzymic reaction favors adenosine 5'-triphosphate synthesis. Thus, a mechanism for adenosine 5'-triphosphate generation in mycoplasmas is suggested.
D000084:Acetate Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000128:Acholeplasma laidlawii / Q000201:enzymology*; D000255:Adenosine Triphosphate / Q000096:biosynthesis; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009180:Mycoplasmatales Infections / Q000382:microbiology*; D010770:Phosphotransferases / Q000378:metabolism*; D013379:Substrate Specificity
D008345:Manganese; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000084:Acetate Kinase; D008274:Magnesium