The kinetic mechanism of S-adenosyl-L-methionine: glutamylmethyltransferase from Salmonella typhimurium.
The kinetic mechanism of the CheR methyltransferase, S-adenosyl-L-methionine (AdoMet): protein-L-glutamate O-methyltransferase (EC 2.1.1.24), from Salmonella typhimurium was investigated. Initial velocity, product inhibition, and binding studies were performed, and from the data obtained, it was determined that the mechanism of the reaction catalyzed by the enzyme is random. Initial velocity rates were measured with varied amounts of both substrates, and double-reciprocal plots gave patterns which converged on or near the abscissa. The products, S-adenosyl-L-homocysteine and methylated receptor, were found to be competitive inhibitors with respect to both AdoMet and receptor. Equilibrium dialysis and immunoprecipitation studies indicated that the two substrates can bind to the enzyme independent of each other. These results are consistent with a random mechanism with no abortive complexes being formed. The Michaelis constants calculated for AdoMet and receptor were 8.62 microM and 2.03 mg/ml total membrane protein (approximately 2.10 microM Tar protein), and the apparent dissociation constants of AdoMet and the receptor were 16.8 microM and 4.07 mg/ml total membrane protein (approximately 4.2 microM Tar protein), respectively. The Kd of AdoMet for the enzyme was 10.9 microM as determined by binding studies.
Simms
S A
SA
Department of Chemistry, City College, New York, New York 10031.
Subbaramaiah
K
K
D000577:Amides / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D002628:Chemoreceptor Cells; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000032:analysis; D011497:Protein O-Methyltransferase / Q000378:metabolism*; D011956:Receptors, Cell Surface; D012486:Salmonella typhimurium / Q000201:enzymology*; D013379:Substrate Specificity
D000577:Amides; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; D008780:Methyltransferases; D011497:Protein O-Methyltransferase