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, 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.