Abstract
Glucokinase (GK) is an important enzyme for regulating blood glucose levels and a potentially attractive target for diabetes of the young type 2 and persistent hyperinsulinemic hypoglycemia of infancy. To characterize the conformational transition of GK from the closed state to the superopen state, a series of conventional molecular dynamics (MD) and target MD (TMD) simulations were performed on both the wild-type enzyme and its mutants. Two 10-ns conventional MD simulations showed that, although the allosteric site of GK is approximately 20 A away from the active site, the activator is able to enhance the activity of the enzyme through conformational restriction. Fourteen TMD simulations on GK and five of its mutants revealed a reliably conformational transition pathway. The overall conformational transition includes three stages, and three likely stable intermediate states were identified by free energy scanning for the snapshots throughout the pathway. The conformational transition feature revealed by our TMD simulations rationalized several important mutagenesis and kinetic data. Remarkably, the TMD simulations predicted that Y61S, I159A, A201R, V203E, and V452S mutations, which have not been investigated so far, may facilitate the opening process of GK. These predictions also have been verified by mutagenesis and kinetic analyses in this study. These observations are beneficial to understanding the mechanism of GK regulation and designing the compounds for treating metabolic diseases.