A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase Export

@article{citeulike:2827114, abstract = {The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod-Wyman-Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R-T equilibrium by using highly deuterated, 1H,13C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR. Although only the T state of the enzyme can be observed in spectra of wild-type unliganded aspartate transcarbamoylase, binding of active-site substrates shift the equilibrium so that correlations from the R state become visible, allowing the equilibrium constant (L') between ligand-saturated R and T forms of the enzyme to be measured quantitatively. The equilibrium constant between unliganded R and T forms (L) also is obtained, despite the fact that the R state is "invisible" in spectra, by means of an indirect process that makes use of relations that emerge from the fact that ligand binding and the R-T equilibrium are linked. Titrations with MgATP unequivocally establish that its binding directly perturbs the R-T equilibrium, consistent with the Monod-Wyman-Changeux model. This study emphasizes the utility of modern solution NMR spectroscopy in understanding protein function, even for systems with aggregate molecular masses in the hundreds of kilodaltons. 10.1073/pnas.0703347104}, author = {Velyvis, Algirdas and Yang, Ying R. and Schachman, Howard K. and Kay, Lewis E.}, citeulike-article-id = {2827114}, citeulike-linkout-0 = {http://dx.doi.org/10.1073/pnas.0703347104}, citeulike-linkout-1 = {http://www.pnas.org/cgi/content/abstract/104/21/8815}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/17502625}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=17502625}, day = {22}, doi = {10.1073/pnas.0703347104}, journal = {Proceedings of the National Academy of Sciences}, keywords = {biggies, dynamics, ligands, methyl, relaxation, trosy}, month = {May}, number = {21}, pages = {8815--8820}, posted-at = {2008-05-23 23:09:44}, priority = {2}, title = {A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase}, url = {http://dx.doi.org/10.1073/pnas.0703347104}, volume = {104}, year = {2007} }

TY - JOUR ID - citeulike:2827114 L3 - citeulike-article-id:2827114 N2 - The 306-kDa aspartate transcarbamoylase is a well studied regulatory enzyme, and it has emerged as a paradigm for understanding allostery and cooperative binding processes. Although there is a consensus that the cooperative binding of active site ligands follows the Monod-Wyman-Changeux (MWC) model of allostery, there is some debate about the binding of effectors such as ATP and CTP and how they influence the allosteric equilibrium between R and T states of the enzyme. In this article, the binding of substrates, substrate analogues, and nucleotides is studied, along with their effect on the R-T equilibrium by using highly deuterated, 1H,13C-methyl-labeled protein in concert with methyl-transverse relaxation optimized spectroscopy (TROSY) NMR. Although only the T state of the enzyme can be observed in spectra of wild-type unliganded aspartate transcarbamoylase, binding of active-site substrates shift the equilibrium so that correlations from the R state become visible, allowing the equilibrium constant (L') between ligand-saturated R and T forms of the enzyme to be measured quantitatively. The equilibrium constant between unliganded R and T forms (L) also is obtained, despite the fact that the R state is "invisible" in spectra, by means of an indirect process that makes use of relations that emerge from the fact that ligand binding and the R-T equilibrium are linked. Titrations with MgATP unequivocally establish that its binding directly perturbs the R-T equilibrium, consistent with the Monod-Wyman-Changeux model. This study emphasizes the utility of modern solution NMR spectroscopy in understanding protein function, even for systems with aggregate molecular masses in the hundreds of kilodaltons. 10.1073/pnas.0703347104 IS - 21 JF - Proceedings of the National Academy of Sciences EP - 8820 TI - A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase VL - 104 SP - 8815 KW - biggies KW - dynamics KW - ligands KW - methyl KW - relaxation KW - trosy AU - Velyvis, Algirdas AU - Yang, Ying AU - Schachman, Howard AU - Kay, Lewis PY - 2007/05/22/ UR - http://dx.doi.org/10.1073/pnas.0703347104 ER -