Lys169 of Human Glucokinase Is a Determinant for Glucose Phosphorylation: Implication for the Atomic Mechanism of Glucokinase Catalysis

摘要

Glucokinase (GK) is an important enzyme for regulating blood glucose levels and an attractive drug target for the maturity-onset diabetes of the young (MODY) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). To characterize the phosphorylation mechanism of glucose catalyzed by GK, molecular modeling, molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations in conjunction with mutagenesis and enzymatic kinetic analysis were performed on both the wild-type enzyme and its mutant. Our threedimensional (3D) model of the GK-Mg2+-ATP-glucose (GMAG) complex showed that GK provides a favorable microenvironment for the phosphorylation of glucose, which is in agreement with a large number of mutagenesis analysis data. Remarkably, a 10-ns MD simulation revealed that Lys169, a naturally occurring mutation (K169N) in the MODY, plays a dominant role in glucose phosphorylation. This prediction has also been verified by mutagenesis and kinetic analyses in this study. The QM/MM calculations on the mechanisms of the glucose phosphorylation catalyzed by both GK and GKK19A mutant further uncover the functional roles of Lys169 to metabolize glucose. In addition to enhancing the binding of GK with both ATP and glucose and bridging ATP and glucose together, Lys169 directly participates in the glucose phosphorylation as a general acid catalyst. These results are beneficial to understanding the catalytic mechanism of GK and the pathogenic mechanism of MODY, and facilitate to discovering drugs for treating metabolic diseases.