Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase

Binnur Eroglu, Susan G. Powers-Lee

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the γ-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.

Original languageEnglish (US)
Pages (from-to)1-9
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume407
Issue number1
DOIs
StatePublished - Nov 18 2002
Externally publishedYes

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Carbamyl Phosphate
Hand Strength
Ligases
Yeast
Saccharomyces cerevisiae
Adenosine Triphosphate
Mutation
Mutagenesis
Ribose
Adenine
Site-Directed Mutagenesis
Amides
Phosphates

Keywords

  • ATP
  • ATP-grasp
  • Amidotransferase
  • Arginine
  • Carbamoyl phosphate
  • Carbamoyl phosphate synthetase
  • Glutamine
  • Pyrimidine
  • Urea

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase. / Eroglu, Binnur; Powers-Lee, Susan G.

In: Archives of Biochemistry and Biophysics, Vol. 407, No. 1, 18.11.2002, p. 1-9.

Research output: Contribution to journalArticle

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