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.1 The Michaelis constants for NH 3 , MgATP and HCO − 3 were 0.7, 1.2 and 2 mM respectively. N ‐Acetyl‐glutamate activated the enzyme with a K a of about 0.1 mM. At sturating concentrations of substrates and effectors the enzyme was inhibited by 50% by carbamoyl phosphate at a concentration of 13 mM. 2 Binding of N ‐acetylglutamate to carbamoyl‐phosphate synthetase required the presence of both free Mg 2+ ions and MgATP, and was inhibited by Ca 2+ ions and by N ‐carbamoylglutamate. The known activation of carbamoyl‐phosphate synthetase by free Mg 2+ is due to an increased affinity of the enzyme for N ‐acetylglutamate. 3 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. 4 Dissociation of N ‐acetylglutamate from the enzyme was relatively fast, with a half‐time of about 5 min. 5 Under all conditions studies there was a close relationship between carbamoyl‐phosphate synthetase activity and the amount of N ‐acetylglutamate bound to the enzyme. 6 The data are discussed in relation to the control of carbamoyl‐phosphate synthetase in the intact hepatocyte.