DIFFERENTIAL ROLES FOR 3 CONSERVED HISTIDINE-RESIDUES WITHIN THE LARGE SUBUNIT OF CARBAMOYL PHOSPHATE SYNTHETASE

Three conserved histidine residues, His-243, His-781, and His-788, located within the large subunit of carbamoyl phosphate synthetase from Escherichia coli were identified by sequence identity comparisons. These three histidine residues were individually mutated to asparagine residues. The H243N mutant enzyme was found to bc critical for carbamoyl phosphate synthesis as the mutant protein was unable to synthesize carbamoyl phosphate at a significant rate (<1/1500). By analysis of the effects of this mutation on the partial reactions catalyzed by CPS, it was determined that this mutation blocked the formation of the carbamate intermediate from carboxyphosphate and ammonia. The H781N mutant enzyme had an order of magnitude reduction for both the rate of carbamoyl phosphate formation and ATP synthesis which is consistent with the proposal that the carboxyl-terminal half of the large subunit is primarily involved in the phosphorylation of the putative carbamate intermediate. This mutation also reduced the effects of the allosteric activator ornithine on the K(m) parameters for ATP in the overall biosynthetic reaction and ADP in the ATP synthesis reaction. The H788N mutant enzyme is a functional protein which maintains the ability to synthesize carbamoyl phosphate at a rate comparable to that of the wild-type enzyme. The effects of this mutation are 10-fold reductions of the ATP synthetase and the bicarbonate-dependent ATPase activities with substantial increases in the K(m), values for ATP in the full biosynthetic reaction and for ADP in the ATP synthesis reaction. The reaction of the wild-type and mutant enzymes with diethylpyrocarbonate (DEPC) was also investigated. All of the activities of the enzyme are inactivated by treatment with high concentrations of DEPC. For the bicarbonate-dependent ATPase activity of the wild-type H781N and H788N enzymes, treatment with DEPC results in an initial increase in ATPase activity followed by a slower inactivation. This biphasic behavior was not observed with the H243N mutant enzyme.