Abstract
Glucokinase (GK), a glucose sensor, maintains plasma glucose homeostasis via phosphorylation of glucose and is a potential
therapeutic target for treating maturity-onset diabetes of the young (MODY) and persistent hyperinsulinemic hypoglycemia
of infancy (PHHI). To characterize the catalytic mechanism of glucose phosphorylation by GK, we combined molecular
modeling, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations,
experimental mutagenesis and enzymatic kinetic analysis on both wild-type and mutated GK. Our three-dimensional
(3D) model of the GK-Mg2+-ATP-glucose (GMAG) complex, is in agreement with a large number of mutagenesis data, and
elucidates atomic information of the catalytic site in GK for glucose phosphorylation. A 10-ns MD simulation of the GMAG
complex revealed that Lys169 plays a dominant role in glucose phosphorylation. This prediction was verified by
experimental mutagenesis of GK (K169A) and enzymatic kinetic analyses of glucose phosphorylation. QM/MM calculations
were further used to study the role of Lys169 in the catalytic mechanism of the glucose phosphorylation and we found that
Lys169 enhances the binding of GK with both ATP and glucose by serving as a bridge between ATP and glucose. More
importantly, Lys169 directly participates in the glucose phosphorylation as a general acid catalyst. Our findings provide
mechanistic details of glucose phorphorylation catalyzed by GK, and are important for understanding the pathogenic
mechanism of MODY.