Constructing a reporter for the activated conformation of carbamoyl phosphate synthetase

Abstract

Whereas the primary role of most carbamoyl phosphate synthetases (CPSs) is catalysis of the entry step of arginine and/or pyrimidine biosynthesis, using glutamine as a nitrogen source, CPSI has evolved to function in the urea cycle and to use only free ammonia. Additional features unique to CPSI are a requirement for the allosteric activator N-acetylglutamate (AGA) and conserved cysteine residues at positions 1327 and 1337 in human CPSI (hCPSI). Upon AGA activation, the cysteine residues assume a proximate arrangement and can form a disulfide bond in the presence of phenylarsine oxide. Since the only available crystal structure is for the Escherichia coli CPS (eCPS), we are carrying out reciprocal mutation engineering of that enzyme with the goal of using disulfide formation in eCPS as a reporter of the activated conformation. eCPS residues Pro909 and Gly919 correspond, respectively, to hCPSI residues Cys1327 and Cys1337. The individual mutations Pro909Cys and Gly919Cys are tolerated by eCPS. Gly919Cys is able to catalyze carbamoyl phosphate formation nearly as well as wild type eCPS, with respective specific activities of 0.22 and 0.86 μmoles citrulline/min/mg. Pro909Cys has a much lower specific activity of 0.0083 μmoles citrulline/min/mg. Both mutants have significantly decreased ATP Km values, with a 25 fold decrease for Pro909Cys and a 100 fold decrease for Gly919Cys. Analysis of the double mutant Pro909Cys Gly919Cys is underway.