Evidence that carboxyphosphate is a kinetically competent intermediate in the carbamyl phosphate synthetase reaction.

In the bicarbonate-dependent ATPase and carbamyl phosphate synthesis reactions catalyzed by Escherichiu coli carbamyl phosphate synthetase, the Pi formed was found to contain one oxygen derived from bicarbonate. This indicates that the bicarbonate requirement for ATPase activity is due to the same direct involvement of HC03in the ATP cleavage reaction as occurs in the presence of ammonia or glutamine. Kinetic evidence for a carboxyphosphate intermediate in the ATPase reaction was obtained by studying reversible ATP cleavage using fly bridge$ non-bridge positional oxygen exchange in By bridge-labeled [‘*O]ATP. The enzyme was found to catalyze the reversible cleavage of ATP to bound ADP in the presence of bicarbonate and absence of ammonia or glutamine, at a rate that is 1.4 to 1.7 times the rate of net ATP cleavage to free ADP and Pi. These results lend support to earlier chemical evidence for such an intermediate. In the same experiment, bicarbonate oxygen was not incorporated to a measurable extent into the ATP y-POJ. Therefore, the regeneration of ATP does not occur from a complex of the form E l ADP l Pi l CO2 or a form in rapid equilibrium with it. The bicarbonate requirement for the reversible cleavage of ATP suggests that this intermediate con-

• Publication year

  1979

• Venue

  Journal of Biological Chemistry

• Publication date

  1979-03-25

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1016/s0021-9258(17)37734-7PMID 217873
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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