ROLE OF CONSERVED RESIDUES WITHIN THE CARBOXY PHOSPHATE DOMAIN OF CARBAMOYL-PHOSPHATE SYNTHETASE

Carbamoyl phosphate synthetase (CPS) catalyzes the formation of carbam oyl phosphate from glutamine, bicarbonate, and 2 mol of MgATP. The het erodimeric protein is composed of a small amidotransferase subunit and a larger synthetase subunit. The synthetase subunit contains a large tandem repeat for each of the nucleotides used in the overall synthesi s of carbamoyl phosphate. A working model for the three-dimensional fo ld of the carboxy phosphate domain of CPS was constructed on the basis of amino acid sequence alignments and the X-ray crystal structure coo rdinates for biotin carboxylase and D-alanine:D-alanine ligase. This m odel was used to select ten residues within the carboxy phosphate doma in of CPS for modification and subsequent characterization of the kine tic constants for the mutant proteins, Residues R82, R129, R169, D207, E215, N283, and Q285 were changed to alanine residues; residues E299 and R303 to glutamine; and residue N301 to aspartate. No significant c hanges in the catalytic constants were observed upon mutation of eithe r R82 or D207, and thus these residues appear to be nonessential for b inding and/or catalytic activity, The Michaelis constant for ATP was m ost affected by modification of residues R129, R169, Q285, and N301. T he binding of bicarbonate was most affected by the mutagenesis of resi dues E215, E299, N301, and R303. The mutation of residues E215, N283, E299, N301, and R303 resulted in proteins which were unable to synthes ize carbamoyl phosphate at a significant rate. All of the mutations, w ith the exception of the N301D mutant, primarily affected the enzyme b y altering the step for the phosphorylation of bicarbonate. However, m utation of N301 to aspartic acid also disrupted the catalytic step inv olved in the phosphorylation of carbamate. These results are consisten t with a role for the N-terminal half of the synthetase subunit of CPS that is primarily directed at the initial phosphorylation of bicarbon ate by the first ATP utilized in the overall synthesis of carbamoyl ph osphate, The active site structure appears to be very similar to the o nes previously determined for D-alanine:D-alanine ligase and biotin ca rboxylase.