Carbamyl phosphate biosynthesis in Bacillus subtilis

1. 1. Two enzyme system capable of synthesizing carbamyl phosphate in cell-free extracts of Bacillus subtilis were studied after separation from each other by fractionation with (NH 4 2 SO 4 . 2. 2. Carbamyl phosphate synthetase utilizes as substrates ATP ( K m = 4.9 mM), glutamine ( K m = 1.8 mM) and bicarbonate; and has a pH optimum of 7.5. It is inhibited by arginine and pyrimidines, and its formation is repressed by uracil, with a concerted effect of arginine at lower uracil concentrations. A biosynthetic role for this enzyme is thus indicated. 3. 3. Carbamate kinase (ATP:carbamate phosphotransferase, EC 2.7.2.2) uses ATP ( K m = 15 mM) and ammonium carbamate ( K m = 8mM), but not glutamine, as substrates. Its pH optimum is 8.5. It is more stable than carbamyl phosphate synthetase. It is partially inhibited by glutamine and arginine, and induced by arginine. This induction suggests a catabolic role. 4. 4. Single mutational events lead to auxotrophy for arginine and pyrimidines, and marked reductions in the levels of carbamyl phosphate synthetase and carbamate kinase. The double requirement can be most simply understood if a single synthetase feeds into a carbamyl phosphate pool common to the arginine and the pyrimidine pathways. The pleiotropic effect on the two enzyme systems points to additional interrelationships. Perhaps the two enzymes share structural or regulatory elements.