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Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism

Sternisha, Shawn M., Liu, Peilu, Marshall, Alan G., Miller, Brian G.
Biochemistry 2018 v.57 no.10 pp. 1632-1639
active sites, enzyme activation, gain-of-function mutation, glucokinase, glucose, humans, hyperinsulinemia, mass spectrometry, viscosity
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.