Mechanism of mitochondrial carbamoyl-phosphate synthetase. Synthesis and properties of active carbon dioxide, precursor of carbamoyl phosphate

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Abstract
The formation of active CO2 (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase is demonstrated. Absence of NH3 is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. ATP and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 [1/2 life] = 0.75 s at 24.degree. C at neutral pH). In the absence of NH3, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The apparent Km are approximately equal to those found for the ATPase activity of the enzyme. The maximum level of CO2-P is limited by the amount of enzyme, and approximates 4 mol of intermediate/mol of enzyme. The unprotonated form of NH3 seems to be the species reacting with CO2-P to produce CP. The reaction of CO2-P and NH3 is very fast (rate constant kn = 8 .times. 104 M-1 s-1) and does not consume free ATP. Therefore, the 2 mol of ATP necessary for CP synthesis binds or reacts with the enzyme and/or CO2 prior to reaction with NH3. The reaction of CO2-P with NH3 also takes place in acetone under conditions at which the enzyme is not active, suggesting little or no assistance from enzyme catalysis or that a part of the catalytic site is frozen by the solvent in the active conformation. In the light of these and other findings, a new scheme is proposed for the mechanism of frog liver carbamoyl-phosphate synthetase and some considerations are made on the chemical nature of the intermediate and on the possible evolutionary significance of the reaction of CO2-P with NH3 in acetone.