Quantitative NMR studies of high molecular weight proteins: Application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G

A high-resolution multidimensional NMR study of ligand-binding to Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is presented. MSG catalyzes the condensation of glyoxylate with an acetyl group of acetyl-CoA, producing malate, an intermediate in the citric-acid cycle. We show that despite the size of the protein, important structural and dynamic information about the molecule can be obtained on a per-residue basis. N-15-(HN)-H-1 residual dipolar couplings and carbonyl chemical shift changes upon alignment in Pf1 phage establish that there are no significant domain reorientations in the molecule upon ligand binding, in contrast to what was anticipated on the basis of both the X-ray structure of the glyoxylate-bound form of the enzyme and structural studies of a related set of proteins. The chemical shift changes of (HN)-H-1, N-15 and (CO)-C-13 nuclei upon binding of pyruvate, a glyoxylate-mimicking inhibitor, and acetyl-CoA have been mapped onto the three-dimensional structure of the molecule. Binding constants of pyruvate, glyoxylate, and acetyl-CoA (in the presence of pyruvate) have been measured, along with the kinetic parameters for glyoxylate and pyruvate binding. The on-rates of pyruvate and glvoxalate binding, similar to1.2 X 10(6) M-1 s(-1) and similar to2.7 X 10(6)M(-1) s(-1), respectively, are significantly lower than what is anticipated from a simple diffusion-controlled process. Some structural implications of the chemical shift perturbations upon binding and the estimated ligand on-rates are discussed. (C) 2003 Elsevier Science Ltd. All rights reserved.