Comparison of the functional differences for the homologous residues within the carboxy phosphate and carbamate domains of carbamoyl phosphate synthetase.

Published

Journal Article

Carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from two molecules of MgATP, bicarbonate, and glutamine. It has been previously shown that the amino- and carboxy-terminal halves of the large subunit of this protein are homologous. 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-dimensional structures of two mechanistically related proteins, biotin carboxylase and D-alanine:D-alanine ligase. The model was tested by mutation of ten amino acid residues predicted to be important for binding and/or catalysis. The mutated residues were as follows: R571, R675, R715, 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, N843D, and R845Q were diminished in their ability to synthesize carbamoyl phosphate. The R715A, Q829A, and R675A mutants displayed elevated Michaelis 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 or 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 phosphorylation of the carbamate intermediate during the final formation of carbamoyl phosphate. The measured effects on the catalyic activities displayed by these mutations were found to be comparable to the previously determined 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) Biochemistry 34, 2768-2776].

Full Text

Duke Authors

Cited Authors

  • Javid-Majd, F; Stapleton, MA; Harmon, MF; Hanks, BA; Mullins, LS; Raushel, FM

Published Date

  • November 12, 1996

Published In

Volume / Issue

  • 35 / 45

Start / End Page

  • 14362 - 14369

PubMed ID

  • 8916923

Pubmed Central ID

  • 8916923

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi961184q

Language

  • eng

Conference Location

  • United States