Properties of carbamoyl-phosphate synthetase (ammonia) in rat-liver mitochondria made permeable with toluene.
Some properties of carbamoyl-phosphate synthetase (ammonia) were studied in rat-liver mitochondria made selectively permeable by pretreatment with toluene. The Michaelis constants for NH3, MgATP and HCO-3 were 0.7, 1.2 and 2 mM respectively. N-Acetylglutamate activated the enzyme with a Ka of about 0.1 mM. At saturating concentrations of substrates and effectors the enzyme was inhibited by 50% by carbamoyl phosphate at a concentration of 13 mM. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase required the presence of both free Mg2+ ions and MgATP, and was inhibited by Ca2+ ions and by N-carbamoylglutamate. The known activation of carbamoyl-phosphate synthetase by free Mg2+ is due to an increased affinity of the enzyme for N-acetylglutamate. Binding of N-acetylglutamate to carbamoyl-phosphate synthetase was a slow process: at N-acetylglutamate concentrations below 0.5 mM maximal binding was not completed within 30 min. The rate of binding increased with increasing N-acetylglutamate concentrations. Dissociation of N-acetylglutamate from the enzyme was relatively fast, with a half-time of about 5 min. Under all conditions studied there was a close relationship between carbamoyl-phosphate synthetase activity and the amount of N-acetylglutamate bound to the enzyme. The data are discussed in relation to the control of carbamoyl-phosphate synthetase in the intact hepatocyte.
D000818:Animals; D001665:Binding Sites; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D005971:Glutamates / Q000378:metabolism; D007700:Kinetics; D008025:Ligases / Q000302:isolation & purification*; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D010539:Permeability; D051381:Rats; D011919:Rats, Inbred Strains; D014050:Toluene / Q000494:pharmacology*
D005971:Glutamates; D014050:Toluene; D008025:Ligases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid