Mechanism of mitochondrial carbamoyl-phosphate synthetase: synthesis and properties of active CO2, precursor of carbamoyl phosphate.
This paper demonstrates the formation of "active CO2" (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase. Absence of ammonia is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. Adenosine triphosphate (ATP) and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 = 0.75 s at 24 degrees C at neutral pH). In the absence of ammonia, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The "apparent KM'S" are approximately equal to those found for the adenosine triphosphate (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 ammonia 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 x 10(4) 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.
D000096:Acetone; D000227:Adenine Nucleotides / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001001:Anura; D001639:Bicarbonates / Q000378:metabolism; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D002221:Carbamyl Phosphate / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism*; D055598:Chemical Phenomena; D002621:Chemistry; D005971:Glutamates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008954:Models, Biological; D010710:Phosphates / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism*; D011892:Rana catesbeiana
D000227:Adenine Nucleotides; D001639:Bicarbonates; D005971:Glutamates; D010710:Phosphates; D000096:Acetone; D002245:Carbon Dioxide; D002221:Carbamyl Phosphate; D000641:Ammonia; D010770:Phosphotransferases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D008274:Magnesium