Carbonic-phosphoric anhydride (carboxy phosphate). Significance in catalysis and regulation of glutamine-dependent carbamyl phosphate synthetase.

Carbonic-phosphoric anhydride (carboxy phosphate) is formed on the enzyme when glutamine-dependent carbamyl phosphate synthetase (Escherichia coli) is incubated with ATP, Mg’+, and HCO,,-. Carboxy phosphate may be trapped by borohydride reduction to formate and as the trimethyl derivative by treatment with diazomethane. The rate of enzyme-bound carboxy phosphate formation is evidently extremely rapid, apparently instantaneous. The total amount of carboxy phosphate trapped on a given amount of enzyme is increased by reagents (L-2-amino-4-oxo-5-chloropentanoate, cyanate) that increase the rate of the HCOamdependent ATPase activity catalyzed by the enzyme, and is inhibited by the cu$-methylene analogs of ATP and ADP which also inhibit the ATPase. Under optimal conditions, the amount of intermediate trapped is not far from stoichiometric with the enzyme. The enzyme does not catalyze ADP-ATP or P,-ATP exchange in the absence of glutamine, suggesting that ADP is a component of the enzyme.carboxy phosphate complex. The effect of allosteric agents on the total amount of carboxy phosphate trapped as formate, and other findings, suggest that carboxy phosphate is bound most efficiently by an intermediate oligomer of the enzyme, probably the dimer. However, the accumulated data indicate that selfassociation is not required for allosteric activation. Previous studies (Trotta, P. P., Pinkus, L. M., Haschemeyer, R. H., and Meister, A. (1974) J. Biol. Chem. 249, 482-491) showed that the monomeric unit of carbamyl phosphate synthetase (M, 163,000) exists in an 8.7 S form in phosphate buffer and in a 7.3 S form in Tris and barbital buffers. This buffer-dependent conformational change is shown here to be associated with changes in the catalytic properties of the first ATP site, i.e. that involved in bicarbonate activation, but apparently does not affect the catalytic properties of the second ATP site, i.e. that involved in phosphorylation of carbamate. In phosphate buffer the bicarbonate-dependent ATPase activity is decreased and sensitized to allosteric effecters. The present and earlier