COMPARISON OF THE FUNCTIONAL DIFFERENCES FOR THE HOMOLOGOUS RESIDUES WITHIN THE CARBOXY PHOSPHATE AND CARBAMATE DOMAINS OF CARBAMOYL-PHOSPHATE SYNTHETASE

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes t he formation of carbamoyl phosphate from two molecules of MgATP, bicar bonate, and glutamine. It has been previously shown that the amino- an d carboxy-terminal halves of the large subunit of this protein are hom ologous. A working model for the active site structure of the carboxy- terminal domain of the large subunit of CPS was constructed based upon amino acid sequence alignments and the previously determined three-di mensional structures of two mechanistically related proteins, biotin c arboxylase and D-alanine:D-alanine ligase. The model was tested by mut ation of ten amino acid residues predicted to be important for binding and/or catalysis. The mutated residues were as follows: R571, R675, R 715, D753, E761, N827, Q829, E841, N843, and R845. The mutant proteins were expressed, purified to homogeneity and the catalytic properties determined for a variety of assay formats. The mutants E761A, E841Q, N 843D, and R845Q were diminished in their ability to synthesize carbamo yl phosphate. The R715A, Q829A, and R675A mutants displayed elevated M ichaelis constants for MgADP in the partial back reaction. The mutants E761A, N827A, E841Q, N843D, and R845Q showed significant increases in the Michaelis constants for either bicarbonate or carbamoyl phosphate . No significant alterations were noted upon mutation of either R571 o r D753 to an alanine residue and thus these amino acids do not appear essential for structure or catalytic activity. These results have been utilized to further support the proposal that the C-terminal half of the large subunit of CPS is primarily responsible for the phosphorylat ion of the carbamate intermediate during the final formation of carbam oyl phosphate. The measured effects on the catalyic activities display ed by these mutations were found to be comparable to the previously de termined effects after mutation of the homologous residues located on the N-terminal half of CPS and also for those residues mutated within D-alanine:D-alanine ligase [Shi, Y., & Walsh, C. T. (1995) Biochemistr y 34, 2768-2776].