Purification and properties of a kinase from Escherichia coli K-12 that phosphorylates two periplasmic transport proteins.
The phosphorylation in vivo and in vitro of the arginine-ornithine and the lysine-arginine-ornithine (LAO) periplasmic transport proteins of Escherichia coli K-12 was previously reported (Celis, R. T. F. (1984) Eur. J. Biochem. 145, 403-411). The phosphorylative reaction required ATP (as a direct energy donor), Mg2+, and a kinase that can be released by osmotic shock treatment of the cells. The enzyme was purified to electrophoretic homogeneity. The enzyme exhibited an ATPase activity and a kinase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave an apparent molecular weight of 43,000 for the enzyme. The native protein showed the same molecular weight, suggesting that the protein is a monomer. The protein showed an apparent isoelectric point of 4.8 on isoelectric focusing. The two enzymatic reactions required a divalent cation and the apparent Km value for Mg2+ for the kinase activity was 0.5 mM. Mn2+ and Co2+ served as well as Mg2+, whereas Zn2+ and Ca2+ did not support activity. The ATPase activity of the enzyme yielded an apparent Km value for ATP of 50 microM. A similar value, Km of 100 microM, was calculated for the kinase activity with different concentrations of ATP. The enzyme showed a pH optimum of 7.3.
Urban
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Department of Microbiology, New York University School of Medicine, New York 10016.
Celis
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism*; D002845:Chromatography; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009952:Ornithine / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011489:Protein Denaturation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008670:Metals; D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D001120:Arginine; D009952:Ornithine; D011494:Protein Kinases; D008239:Lysine