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 language | English (US) |
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Pages (from-to) | 1-9 |
Number of pages | 9 |
Journal | Archives of Biochemistry and Biophysics |
Volume | 407 |
Issue number | 1 |
DOIs | |
State | Published - 2002 |
Externally published | Yes |
Keywords
- ATP
- ATP-grasp
- Amidotransferase
- Arginine
- Carbamoyl phosphate
- Carbamoyl phosphate synthetase
- Glutamine
- Pyrimidine
- Urea
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Molecular Biology