The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry.
Differential scanning calorimetry of Escherichia coli carbamoyl-phosphate synthetase and its isolated large and small subunits reveals in each case an irreversible, kinetically controlled transition, at a temperature 14 degrees C higher for the holoenzyme than for the subunits, indicating dramatic stabilization of the subunits in the heterodimer. The deletion of the COOH-terminal 171 (mutant CarB'2373) or 385 (mutant CarB2177) residues of the large subunit results in more asymmetric transitions at a temperature 7 degrees C lower than for the wild type. The allosteric effectors IMP, UMP, and ornithine induce small reversible transitions at low temperature in the endotherm for the wild-type enzyme, but not for CarB'2373, as expected if the effectors bind in the 171-residue, COOH-terminal region. In contrast, two ligands that bind outside the deleted region, Ap5A (a ligand of both ATP sites) and glycine (an analog of glutamine) decrease and increase, respectively, the stability of the two mutants and of the wild type. The stabilization by glycine requires that the subunits are associated. The results support the implication of the 20-kDa COOH-terminal domain of the large subunit in the allosteric modulation by all the effectors and are consistent with the folding of the large subunit as a pseudohomodimer of its two homologous halves.
Cervera
J
J
Instituto de Investigaciones Citológicas de la Fundación Valenciana de Investigaciones Biomédicas (Centro Asociado del CSIC), Spain.
Conejero-Lara
F
F
Ruiz-Sanz
J
J
Galisteo
M L
ML
Mateo
P L
PL
Lusty
C J
CJ
Rubio
V
V
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D003062:Codon; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D005998:Glycine / Q000494:pharmacology; D007291:Inosine Monophosphate / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009952:Ornithine / Q000494:pharmacology; D017510:Protein Folding; D014542:Uridine Monophosphate / Q000494:pharmacology
D003062:Codon; D046911:Macromolecular Substances; D007291:Inosine Monophosphate; D014542:Uridine Monophosphate; D009952:Ornithine; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D005998:Glycine