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 gamma-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.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Adenosine Triphosphate / metabolism*
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Amino Acid Sequence
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Binding Sites
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Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)*
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Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / chemistry
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Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / genetics*
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Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / metabolism*
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DNA Mutational Analysis
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Fungal Proteins / chemistry
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Fungal Proteins / genetics
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Fungal Proteins / metabolism
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Protein Folding
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics*
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Saccharomyces cerevisiae Proteins / metabolism*
Substances
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Fungal Proteins
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Saccharomyces cerevisiae Proteins
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Adenosine Triphosphate
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Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor
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carbamoyl phosphate synthetase (arginine-specific)
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CPA1 protein, S cerevisiae
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Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)