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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.allostery ͉ Monod-Wyman-Changeux (MWC) model ͉ NMR spectroscopy ͉ methyl-transverse relaxation optimized spectroscopy ͉ ligand binding Q uantitative, site-specific studies of proteins by NMR spectroscopy for the most part have been restricted to systems with molecular masses on the order of 50 kDa or less. With the development of new labeling schemes along with experiments that optimally preserve NMR signals, it now has become possible to investigate much larger complexes (1). For example, Wüth-rich, Horwich, and coworkers have used 1 H-15 N cross-correlated relaxation-induced polarization transfer (CRIPT) spectroscopy to establish which residues of highly deuterated GroES interact with GroEL in a GroES-GroEL complex that is 900 kDa (2) and to study interactions between substrates and GroEL (3). Sprangers and coworkers have exploited methyl-transverse relaxation optimized spectroscopy (TROSY) of 1 H, 13 C-methyl-labeled probes in the 300-kDa highly deuterated protease ClpP to quantify dynamics and relate it to function (4). In a second study, this methyl-based approach has been used to measure sitespecific dynamics in the 20S proteasome (670 kDa) along with binding to target molecules (5).The methodology that now is available opens the possibility for the study of a wide range of molecular machines in a site-specific...

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