title: A solution NMR study showing that active site ligands and nucleotides directly perturb the allosteric equilibrium in aspartate transcarbamoylase.
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, (1)H,(13)C-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.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8.
authors.forename: Algirdas
authors.initials: A
authors.lastname: Velyvis
authors.forename: Ying R
authors.initials: YR
authors.lastname: Yang
authors.forename: Howard K
authors.initials: HK
authors.lastname: Schachman
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
mesh_terms: D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D008024:Ligands; D008745:Methylation; D019906:Nuclear Magnetic Resonance, Biomolecular; D009711:Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D017186:Titrimetry
chemical_list: D008024:Ligands; D009711:Nucleotides; D001221:Aspartate Carbamoyltransferase