Studies on the function of N-acyl glutamates in the carbamyl phosphate synthetase reaction

Studies were undertaken to investigate whether the natural co-factor of carbamyl-P synthetase, acetylglutamate, could act as a carrier for carbonate and carboxy-phosphate during carbamyl-P synthesis, as suggested earlier by Jones and Spector (1). For acetylglutamate to so act, either the α-amide N must lose a hydrogen or the carbonyl oxygen must enolize, which would result in a loss of a hydrogen from either the nitrogen or the acyl-methyl group. One approach to this problem is synthesis of appropriate analogues of acetylglutamate followed by a test of their ability to act as co-factor. Schooler et al. (2) have reported that 2-acetoxyglutarate is an effective co-factor for the enzyme, indicating that two of the above alternatives were unlikely. Their work is not, as yet, definitive and therefore requires confirmation, for they have not reported the method of synthesis used to prepare 2-acetoxyglutarate nor its complete characterization. This paper reports these details and confirms their observation that 2-acetoxyglutarate is an effective cofactor. In addition the ability of mono- and trifluoroacetyl glutamate to serve as co-factor is reported. A fourth analogue, 3-acetoxyglutarate will not serve as a cofactor. The third alternative i.e., enolization of the carbonyl group by the loss of a hydrogen atom of the acetyl-methyl group, was tested by experiments where the solvent was TOH. These experiments indicate that no hydrogen is labilized from the acetyl-methyl group. These results and earlier studies from a number of laboratories have completely tested the functional groups of active co-factors and consistently show that an active co-factor does not react with the other substrates (ammonia, bicarbonate, and ATP) required for carbamyl-P synthesis by carbamyl-P synthetase.