Identification of enzyme-bound activated CO2 as carbonic-phosphoric anhydride: isolation of the corresponding trimethyl derivative from the active site of glutamine-dependent carbamyl phosphate synthetase.
The activated CO2 intermediate formed in the reaction catalyzed by glutamine-dependent carbamyl phosphate synthetase was identified as carbonic-phosphoric anhydride through the use of two independent procedures. The carboxy phosphate intermediate was reduced to formate by treatment with potassium borohydride. Although both free CO2 and the enzyme-bound activated CO2 are reduced to formic acid by borohydride, it was possible to selectively introduce a 14C label into the enzyme-bound activated CO2 and thus into the formic acid derived from it. Such [14C]formate formation required the presence of ATP, KCl, and the enzyme, and evidence was obtained that the [14C]formate found is not derived from carbamyl phosphate or from bicarbonate bound nonspecifically to the enzyme. When the enzyme was treated with L-2-amino-4-oxo-5-chloropentanoate (or cyanate), the formation of [14C]formate was increased about 2-fold, a finding consistent with the previous observation that such treatment effects a similar increase in the bicarbonate-dependent cleavage of ATP catalyzed by the enzyme. When reaction mixtures containing the enzyme, [gamma-32P]ATP, and [14C]bicarbonate were methylated by treatment with diazomethane, a labeled compound was formed which cochromatographed with authentic trimethyl carboxy phosphate. Equimolar quantities of 14C and 32P wer incorporated into the intermediate, thus confirming its identification as carboxy phosphate. Nonenzymatic transphosphorylation from ATP to bicarbonate to form carboxy phosphate was also detected by diazomethane trapping.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000812:Anhydrides / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D010755:Organophosphates; D009943:Organophosphorus Compounds; D010770:Phosphotransferases / Q000378:metabolism*
D000812:Anhydrides; D001639:Bicarbonates; D010755:Organophosphates; D009943:Organophosphorus Compounds; C530383:trimethyl carboxyphosphate; D002245:Carbon Dioxide; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)