Limited proteolysis reveals low-affinity binding of N-acetyl-L-glutamate to rat-liver carbamoyl-phosphate synthetase (ammonia).
Carbamoyl-phosphate synthetase was inactivated by elastase with first-order kinetics, and N-acetyl-L-glutamate speeded inactivation. From the dependence of the t1/2 value for inactivation on the concentration of acetylglutamate we estimate a Kd value for binding of the activator of 0.365 mM, which is approximately 600 times greater than in the presence of ATP, HCO3-, K+ and Mg2+. K+ and Mg2+ are not required for binding with low affinity, and in the absence of ATP they do not appear to increase the affinity for acetylglutamate. In the presence of acetylglutamate, mixtures of ATP, K+ and Mg2+ protect the enzyme from inactivation. ADP or AdoPP[NH]P partly replaced ATP in protecting the enzyme and thus binding of the nucleotide without further reaction is enough for protection. Two partial activities of the enzyme were inactivated by elastase to the same extent as the overall reaction, and thus elastase affects some property of the enzyme which is essential for catalysis. With other proteinases tested, inactivation was also accelerated by acetylglutamate and was slowed by mixtures of ATP, K+, Mg2+ and acetylglutamate, suggesting that changes in the accessibility of susceptible bonds are responsible for the changes in the degree of inactivation. It is concluded that elastase attacks at or close to the binding sites for ATP, and that exposure of the binding site for the ATP molecule that yields Pi (ATPA) upon binding of acetylglutamate causes the acceleration of the proteolytic inactivation.
D000818:Animals; D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008954:Models, Biological; D010196:Pancreatic Elastase / Q000494:pharmacology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D051381:Rats
D005971:Glutamates; D010446:Peptide Fragments; D010196:Pancreatic Elastase; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid