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-phosph ate synthetase and its isolated large and small subunits reveals in ea ch case an irreversible, kinetically controlled transition, at a tempe rature 14-degrees-C higher for the holoenzyme than for the subunits, i ndicating dramatic stabilization of the subunits in the heterodimer. T he deletion of the COOH-terminal 171 (mutant CarB'2373) or 385 (mutant CarB2177) residues of the large subunit results in more asymmetric tr ansitions at a temperature 7-degrees-C lower than for the wild type. T he allosteric effectors IMP, UMP, and ornithine induce small reversibl e transitions at low temperature in the endotherm for the wild-type en zyme, 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 glyc ine (an analog of glutamine) decrease and increase, respectively, the stability of the two mutants and of the wild type. The stabilization b y glycine requires that the subunits are associated. The results suppo rt the implication of the 20-kDa COOH-terminal domain of the large sub unit in the allosteric modulation by all the effectors and are consist ent with the folding of the large subunit as a pseudohomodimer of its two homologous halves.