Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase.
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.
Eroglu
Binnur
B
Department of Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, USA.
Powers-Lee
Susan G
SG
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C064716:carbamoyl phosphate synthetase (arginine-specific); C403521:CPA1 protein, S cerevisiae; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)