On the Mechanism of Carbamoylphosphate Synthetase

Abstract

Studies were undertaken to investigate whether 2‐acetamido glutaric anhydride is an intermediate in the synthesis of carbamoylphosphate. Experiments were carried out with pools of 2‐acetamido‐glutaric anhydride, its hydroxamate and NH 2 OH to trap the anhydride. The anhydride was also tested as an inhibitor. The experiments show no evidence for the participation of free 2‐acetamido‐glutaric anhydride as an intermediate. The effect of NH 2 OH on the synthesis of ATP from carbamoylphosphate and ADP was investigated in order to clarify whether this reaction is an integral part of the overall reaction catalyzed by carbamoylphosphate synthetase and whether there is a common intermediate which could react with NH 2 OH. The unexpected observation was made that NH 2 OH is an inhibitor of ATP synthesis at low levels of ADP, and that the enzyme has a high affinity for ADP. These results together with other experiments on carbamoylphosphate binding by the enzyme point to a competition between ADP site and NH 2 OH. No increase in the release of P i from carbamoylphosphate in the presence of both acetylglutamate and NH 2 OH was observed; therefore the intermediate responsible for the acetylglutamate NH 2 OH‐dependent ATPase is not formed extensively from carbamoylphosphate. The activation of carbamoylphosphate synthetase by acetylglutamate for both the forward and back reactions was studied. The enzyme was activated with acetylglutamate and rapidly separated from the cofactor on Sephadex. There was no evidence for retention of activation, indicating a fast return to the non‐activated state. Therefore, enzyme samples were activated, diluted and assayed immediately and after time intervals. A time‐dependent reversibility of the activated enzyme for both the forward and back reactions is illustrated.