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

The reaction catalyzed by carbamoyl phosphate synthetase (CPS) from Escheri chia 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 o f ammonia with the carboxy phosphate intermediate or whether the carboxy ph osphate must first dissociate to carbon dioxide prior to the attack by ammo nia. A spectrophotometric pH indicator assay was used to show that during t he bicarbonate-dependent ATPase partial reaction, the initial rate of H+ pr oduction is linear and equivalent to the rate of ADP formation. The time co urse for H+ production is consistent with a mechanism in which carboxy phos phate, bur not CO2, is formed and released from the enzyme. No C-13-NMR sig nal is observed for CO2 during the ATPase reaction at either low or high co ncentration of CPS. Isotope exchange experiments using [gamma-O-18(4)] ATP as the initial substrate in the ATPase reaction produced P-i without the de lectable loss of 18-oxygen, which is inconsistent with the formation of CO2 during the bicarbonate-dependent ATPase reaction. For the partial back rea ction catalyzed by CPS, a pH indicator assay was utilized to test for the i nitial production of carbamate and ATP from carbamoyl phosphate and ADP. Wh en the rate of ATP synthesis was made relatively fast, the time course for H+ production was biphasic. These results are consistent with the rapid upt ake of H+ during the breakdown of carbamate to CO2 and NH4+, followed by th e slower release of protons during the nonenzymatic hydration of CO2. Overa ll, these results are consistent with the formation of carbamate during the partial back reaction of CPS but no evidence could be obtained for the int ermediacy of CO2 during the bicarbonate-dependent ATPase reaction.