Mechanistic Origins Of Enzyme Activation In Human Glucokinase Variants Associated With Congenital Hyperinsulinism

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 alpha-type and beta-type. Steady-state viscosity variation studies indicate that the k(cat), values of wild-type GCK and an alpha-type variant are partially diffusion-limited, whereas the k(cat) value of a beta-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the alpha-type variant display burst kinetics, whereas the beta-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 alpha-type variant. The alpha-type variant also displays an increased level of exchange within a beta-strand located near the enzyme's hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, beta-type activation causes no substantial difference in global or local exchange relative to that of unliganded, wild-type GCK. Together, these results demonstrate that alpha-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas beta-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.