Critical roles for arginine 1061/1060 and tyrosine 1057 in Saccharomyces cerevisiae arginine-specific carbamoyl-phosphate synthetase.
Carbamoyl-phosphate synthetases (CPSases) bind two molecules of ATP at two internally duplicated domains. Previous affinity labeling studies with the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA; Kim, H., Kelly, R. E., and Evans, D. R. (1991) Biochemistry 30, 10322-10329; Potter, M. D., and Powers-Lee, S. G. (1992) J. Biol. Chem. 267, 2023-2031) have identified several peptides as being near the ATP sites, with most of the FSBA-labeled peptides localized to the internally duplicated domains. However, two of the FSBA-labeled peptides were localized to the third domain of CPSase, an autonomously folded but flexible domain at the extreme C-terminus of the protein. These findings suggested that the C-terminal domain is also involved in interaction with both molecules of ATP and that it might serve to complement the ATP binding sites on the duplicated domains by participating in catalytic processing of the ATP molecules. To further define the role of the C-terminal domain in ATP utilization, we have now carried out site-directed mutagenic analysis of peptide 1052-1061 of the Saccharomyces cerevisiae arginine-specific CPSase. Aspartate residues at positions 1053, 1054, and 1056 did not appear to play a significant role in CPSase structure or function. However, tyrosine 1057 was critical for CPSase structure and the presence of one of the tandem arginyl residues at positions 1061 and 1060 was critical for CPSase catalytic function.
Lim
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Department of Biology, Northeastern University, Boston, Massachusetts, 02115, USA.
Powers-Lee
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SG
D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine; D001483:Base Sequence; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D014443:Tyrosine
D014443:Tyrosine; D000255:Adenosine Triphosphate; D001120:Arginine; D008025:Ligases; D019733:Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; C064716:carbamoyl phosphate synthetase (arginine-specific)