Identification of critical amino acid residues of Saccharomyces cerevisiae carbamoyl-phosphate synthetase: definition of the ATP site involved in carboxy-phosphate formation

• Published in: Biochimica et biophysica acta 1997-08, Vol.1341 (1), p.35-48
• Main Authors: Zheng, Weihong, Lim, Angela L, Powers-Lee, Susan G
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Amino Acid Sequence; Amino Acids - analysis; Arginine; ATP; Binding Sites; Carbamoyl-phosphate; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - chemistry; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - genetics; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) - metabolism; Carbamyl Phosphate - metabolism; Escherichia coli - enzymology; Genetic Complementation Test; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - growth & development; Sequence Alignment; Urea; Yeast
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(97)00058-7

Carbamoyl-phosphate synthetases (CPSases) utilize two molecules of ATP at two homologous domains, B and C, with ATP B used to form the enzyme-bound intermediate carboxy-phosphate and ATP C used to phosphorylate the carbamate intermediate. To further define the role of one CPSase peptide suggested by affinity labeling studies to be near the ATP B site, we have carried out site-directed mutagenic analysis of peptide 234–242 of the Saccharomyces cerevisiae arginine-specific CPSase. Mutants E234A, E234D, E236A, E236D and E238A were unable to complement the CPSase-deficient yeast strain LPL26 whereas mutants Y237A, E238D, R241K, R241E and R241P supported LPL26 growth as well as wild-type CPSase. Kinetic analysis of E234A and Y237A indicated impaired utilization of ATP B but not of ATP C. D242A, a temperature-sensitive mutant, retained no detectable activity when assayed in vitro. These findings, together with the affinity labeling data and primary sequence analysis, strongly suggest that the yeast CPSase peptide 234–242 is located at the ATP B site and that some of its residues are important for functioning of the enzyme. D242 appears to occupy a critical structural position and E234, E236 and E238 appear to be critical for function, with the spatial arrangement of the carboxyl side chain also critical for E234 and E236.