PMID:12359710
| Citation |
West, JM, Tsuruta, H and Kantrowitz, ER (2002) Stabilization of the R allosteric structure of Escherichia coli aspartate transcarbamoylase by disulfide bond formation. J. Biol. Chem. 277:47300-4 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1074/jbc.M209913200 |
| Keywords |
Adenosine Triphosphate/pharmacology; Alanine/chemistry; Allosteric Site; Aspartate Carbamoyltransferase/chemistry; Aspartate Carbamoyltransferase/genetics; Aspartic Acid/chemistry; Catalysis; Catalytic Domain; Cross-Linking Reagents; Cysteine/chemistry; Cytidine Triphosphate/pharmacology; Disulfides; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Kinetics; Models, Molecular; Mutation; Protein Binding; Protein Structure, Quaternary; Scattering, Radiation; X-Rays |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.