Phosphorylation of Carbamate in Vivo and in Vitro

The isolation of one-step mutants, simultaneously requiring arginine and uracil for growth1 (Z-6), has indicated the existence of a specific intermediate shared by the arginine- and the pyrimidine-biosynthetic pathways. Biochemical studies have shown that these two pathways do indeed share a common intermediate, carbamyl phosphate (7-9), the synthesis of which from carbon dioxide, ammonia, and adenosine triphosphate seems to depend on only one enzymatic reaction, the phosphorylation of carbamate (10) ; carbamate itself probably is synthesized nonenzymatically (10). It was thus suggested that the lack of the ability to phosphorylate carbamate may explain the double requirement of the one-step mutants2 (5, 6, 12). However, extracts from many of these mutants, isolated from different strains of Escherichia coli in several laboratories, were found to possess a carbamate-phosphorylating activity comparable in amount to that found in the wild-type extracts2 (4-6, 12). This finding is inconsistent with the interpretation that in E. coli the enzymatic synthesis of carbamyl phosphate is the only step in common for the arginine- and the uridine-synthetic pathways. It is possible that in the biosynthesis of carbamate there is another specific common step, but all attempts to demonstrate a difference in the amount of carbamate formed, enzymatically or nonenzymatically, in the presence of extracts of the wild type or of these mutants, have thus far been unsuccessful. An analogous difficulty arises in the interpretation of results obtained with another class of E. coli mutants having the following behavior: in the absence of uracil their growth is independent of, or only stimulated by, arginine; however, in the presence of uracil these cells manifest a strict requirement for arginine or citrulline (13, 14). This observation would be explained if uracil were repressing the synthesis of carbamyl phosphokinase (ATP : carbamate phosphotransferase, EC 2.7.2.2). However, the finding that the extracts of these cells possess a carbamatephosphorylating activity comparable in amount to that found in the wild type, and in the double-requiring mutants described above, is inconsistent with this interpretation (14).