title: Stabilization of the R allosteric structure of Escherichia coli aspartate transcarbamoylase by disulfide bond formation.
abstract: Here we report the first use of disulfide bond formation to stabilize the R allosteric structure of Escherichia coli aspartate transcarbamoylase. In the R allosteric state, residues in the 240s loop from two catalytic chains of different subunits are close together, whereas in the T allosteric state they are far apart. By substitution of Ala-241 in the 240s loop of the catalytic chain with cysteine, a disulfide bond was formed between two catalytic chains of different subunits. The cross-linked enzyme did not exhibit cooperativity for aspartate. The maximal velocity was increased, and the concentration of aspartate required to obtain one-half the maximal velocity, [Asp](0.5), was reduced substantially. Furthermore, the allosteric effectors ATP and CTP did not alter the activity of the cross-linked enzyme. When the disulfide bonds were reduced by the addition of 1,4-dithio-dl-threitol the resulting enzyme had kinetic parameters very similar to those observed for the wild-type enzyme and regained the ability to be activated by ATP and inhibited by CTP. Small-angle x-ray scattering was used to verify that the cross-linked enzyme was structurally locked in the R state and that this enzyme after reduction with 1,4-dithio-dl-threitol could undergo an allosteric transition similar to that of the wild-type enzyme. The complete abolition of homotropic and heterotropic regulation from stabilizing the 240s loop in its closed position in the R state, which forms the catalytically competent active site, demonstrates the significance that the quaternary structural change and closure of the 240s loop has in the functional mechanism of aspartate transcarbamoylase.
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000409:Alanine / Q000737:chemistry; D000495:Allosteric Site; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D003570:Cytidine Triphosphate / Q000494:pharmacology; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation; D014965:X-Rays
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D001224:Aspartic Acid; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D001221:Aspartate Carbamoyltransferase; D003545:Cysteine; D000409:Alanine