Influence of anions on the activation of carbamoyl phosphate synthetase (ammonia) by acetylglutamate: implications for the activation of the enzyme in the mitochondria.
Rat liver carbamoyl phosphate synthetase is shown to be inhibited by anions competitively with acetylglutamate (the allosteric activator of the enzyme) with a potency decreasing in the order NO3- greater than SO4(2-) greater than Cl- approximately HCO3-. Inhibition by chloride accounts for most of the inhibition reported [Lund, P., and Wiggins, D. (1987) Biochem. J. 243, 273-276] in Tris buffer. Mes, acetate, and isethionate give little or no inhibition and phosphate inhibits noncompetitively. Plots of the KA value for acetylglutamate versus the concentration of chloride or nitrate are curved upward and binding assays demonstrate that the inhibitory anions displace acetylglutamate from the enzyme. Thus, the anions may compete with the carboxyls of acetylglutamate for positive charges at the binding site. Of the organic anions found in the mitochondrial matrix, alpha-ketoglutarate, malate, succinate, and citrate increase substantially the KA for acetylglutamate. Changes in the concentrations of ATP, HCO3-, NH4+, and Mg2+, and high concentrations of protein (60 mg/ml serum albumin) influence the KA value. Changes in the concentration of the enzyme have no effect. Under assay conditions approaching the ionic, buffer, and substrate concentrations expected to occur in the mitochondrial matrix, the KA value for acetylglutamate is 27 microM and the Vmax is decreased about 50%. These results indicate that physiological changes in the level of acetylglutamate significantly influence the degree of activation of carbamoyl phosphate synthetase in vivo.
García-España
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A
Laboratory of Cell Chemistry, Instituto de Investigaciones Citológicas de la Caja de Ahorros de Valencia (Centro Asociado del CSIC), Spain.
Alonso
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E
Rubio
V
V
D000818:Animals; D000838:Anions; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D002412:Cations; D004789:Enzyme Activation; D005971:Glutamates / Q000494:pharmacology*; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D012492:Salts
D000838:Anions; D002412:Cations; D005971:Glutamates; D012492:Salts; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid