Requirement for the carboxyl-terminal domain of Saccharomyces cerevisiae carbamoyl-phosphate synthetase.
The arginine-specific carbamoyl phosphate synthetase of Saccharomyces cerevisiae is a heterodimeric enzyme, with a 45-kDa CPA1 subunit binding and cleaving glutamine, and a 124-kDa CPA2 subunit accepting the ammonia moiety cleaved from glutamine, binding all of the remaining substrates and carrying out all of the other catalytic events. CPA2 is composed of two apparently duplicated amino acid sequences involved in binding the two ATP molecules needed for carbamoyl phosphate synthesis and a carboxyl-terminal domain which appears to be less tightly folded than the remainder of the protein. Using deletion mutagenesis, we have established that essentially all of the carboxyl-terminal domain of CPA2 is required for catalytic function and that even small truncations lead to significant changes in the CPA2 conformation. In addition, we have demonstrated that the C-terminal region of CPA2 can be expressed as an autonomously folded unit which is stabilized by specific interactions with the remainder of CPA2. We also made the unexpected finding that, even when ammonia is used as the substrate and there is no catalytic role for CPA1, interaction with CPA1 led to an increase in the Vmax of CPA2 in crude extracts.
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Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
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D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D017931:DNA Primers; D004284:Dogfish; D004926:Escherichia coli / Q000201:enzymology; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017384:Sequence Deletion; D013379:Substrate Specificity
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D005973:Glutamine; D000641:Ammonia; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)