Carbamoyl phosphate synthetase from Escherichia coli does not catalyze the dehydration of bicarbonate to carbon dioxide

The reaction catalyzed by carbamoyl phosphate synthetase (CPS) from Escherichia coli was examined for the formation of several transient intermediates. The chemical mechanism for CPS has been previously postulated to involve the formation of carboxy phosphate and carbamate as transient intermediates. However, it is uncertain whether the carbamate arises from the reaction of ammonia with the carboxy phosphate intermediate or whether the carboxy phosphate must first dissociate to carbon dioxide prior to the attack by ammonia. A spectrophotometric pH indicator assay was used to show that during the bicarbonate-dependent ATPase partial reaction, the initial rate of H+ production is linear and equivalent to the rate of ADP formation. The time course for H+ production is consistent with a mechanism in which carboxy phosphate, bur not CO2, is formed and released from the enzyme. No C-13-NMR signal is observed for CO2 during the ATPase reaction at either low or high concentration of CPS. Isotope exchange experiments using [gamma-O-18(4)] ATP as the initial substrate in the ATPase reaction produced P-i without the delectable loss of 18-oxygen, which is inconsistent with the formation of CO2 during the bicarbonate-dependent ATPase reaction. For the partial back reaction catalyzed by CPS, a pH indicator assay was utilized to test for the initial production of carbamate and ATP from carbamoyl phosphate and ADP. When the rate of ATP synthesis was made relatively fast, the time course for H+ production was biphasic. These results are consistent with the rapid uptake of H+ during the breakdown of carbamate to CO2 and NH4+, followed by the slower release of protons during the nonenzymatic hydration of CO2. Overall, these results are consistent with the formation of carbamate during the partial back reaction of CPS but no evidence could be obtained for the intermediacy of CO2 during the bicarbonate-dependent ATPase reaction.