Carbamoyl‐phosphate synthetase I

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Abstract
The dissociation of the cofactor, acetylglutamate, from the enzyme‐cofactor complex formed by carbamoyl‐phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO−3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s−1) is considerably slower than the rate of enzyme turnover (∼6 s−1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5–2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme‐cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.