In vivo mutational analysis of highly conserved amino acid residues of the small subunit Cpa1p of the carbamylphosphate synthetase of Saccharomyces cerevisiae.
The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site-directed mutagenesis to change all residues to aspartic acid. Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo. The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate. These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site. Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS. C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mM). The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mM, suggesting that this substitution is critical to NH3-dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme.
Bernard
A
A
Laboratoire de Génétique, UPR 9003 CNRS, Institut de Recherche contre les Cancers de l'Appareil Digestif (IRCAD), Hospices Civils, Strasbourg, France.
Erbs
P
P
Demuyter
P
P
Jund
R
R
D000595:Amino Acid Sequence; D000641:Ammonia / Q000378:metabolism; D000645:Ammonium Sulfate / Q000378:metabolism; D001120:Arginine / Q000096:biosynthesis; D001224:Aspartic Acid / Q000235:genetics; D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000378:metabolism*; D002874:Chromosome Mapping; D003545:Cysteine / Q000235:genetics; D015870:Gene Expression; D005972:Glutaminase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011392:Proline / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D017421:Sequence Analysis; D017384:Sequence Deletion; D014170:Transformation, Genetic
D005973:Glutamine; D001224:Aspartic Acid; D006639:Histidine; D000641:Ammonia; D001120:Arginine; D011392:Proline; D005972:Glutaminase; D019731:Carbon-Nitrogen Ligases; C024233:carbamoyl-phosphate synthetase (N-acetylglutamate); D003545:Cysteine; D000645:Ammonium Sulfate