Human
glucokinase (GCK) acts as the body's primary
glucose sensor and plays a critical role in
glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive
enzyme variants that cause
congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and β-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a β-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the β-type variant lacks a burst phase. Comparative
hydrogen-
deuterium exchange mass spectrometry of unliganded
enzymes demonstrates that a disordered active site loop, which folds upon binding of
glucose, is protected from exchange in the α-type variant. The α-type variant also displays an increased level of exchange within a β-strand located near the
enzyme's hinge region, which becomes more
solvent-exposed upon
glucose binding. In contrast, β-type activation causes no substantial difference in global or local exchange relative to that of unliganded, wild-type GCK. Together, these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the
glucose-bound conformation, whereas β-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK's unique kinetic cooperativity.