Abstract
Human glucokinase (GCK) serves as the primary physiological glucose sensor in pancreatic β-cells and hepatocytes, orchestrating systemic glucose homeostasis through cooperative substrate kinetics. Pharmacological targeting of GCK via small-molecule glucokinase activators (GKAs) represents a compelling therapeutic strategy for type 2 diabetes mellitus, yet mechanistic insights into allosteric activation pathways have been hindered by conformational heterogeneity. In this study, we report the structural and biochemical characterization of human GCK in complex with a novel heterocyclic activator, GCKA-714, using single-particle cryogenic electron microscopy (cryo-EM) at a global resolution of 2.38 Å. The high-resolution density map demonstrates that GCKA-714 binds to an allosteric pocket located at the hinge region between the large and small domains, stabilizing the enzyme in its catalytically competent, closed conformation. Biochemical profiling reveals that GCKA-714 substantially decreases the glucose half-saturation constant ($S_{0.5}$) from 8.4 ± 0.3 mM to 2.1 ± 0.1 mM while reducing the Hill coefficient ($n_H$) from 1.72 to 1.08, eliminating kinetic cooperativity. Site-directed mutagenesis targeting key residues within the allosteric pocket (Arg63, Met210, Tyr215, and Val455) abolished or severely attenuated activator responsiveness without impairing intrinsic basal catalytic turnover. Together, these structural and mechanistic insights elucidate the molecular basis of non-essential allosteric modulation in human GCK, providing a rational blueprint for the development of next-generation partial activators that circumvent clinical hypoglycemia.
