Specific 13CH3 Labeling and NMR Reveal the Role of Structural Dynamics to Enzymatic Function

Abstract

To understand the catalytic function of glucokinase, an enzyme that catalyzes glucose during glycolysis following a kinetic cooperative mechanism, we employed 13CH3 labeling of methyl groups and NMR. We showed that the origin of kinetic cooperativity is rooted in intramolecular protein dynamics using kinetic CPMG-NMR data of 17 isoleucine sidechains distributed over all parts of GCK. Residues of glucose-free GCK located in the small domain display a distinct exchange behavior involving multiple conformers that are substantially populated (p > 17%) with a kex = 509 ± 51 s–1, whereas in the glucose-bound form these exchange processes are quenched. This exchange process directly competes with the enzymatic turnover rate at physiological glucose concentrations, thereby generating the sigmoidal rate dependence that defines kinetic cooperativity.