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

Abstract

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 glucose binding, is protected from exchange in the α-type variant. The α-type variant also displays increased 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 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.

Clinical and enzymatic phenotypes in congenital hyperinsulinemic hypoglycemia due to glucokinase‐activating mutations: A report of two cases and a brief overview of the literature

“…Our in vitro studies also showed increased enzyme activities for both mutants, and increased thermal stability for the M197V mutant. A recent report suggests two mechanisms of GCK activation among the gain‐of‐function mutations: 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 production release. It was reported that V91 was located in the allosteric activator region, and as a close neighbor, K90R is likely to be directly activated by structural changes and appears to be α‐type.…”

Molecular and cellular regulation of human glucokinase

“…[79] While the mechanism of action is not known, BAD BH3 domains might stabilize specific conformations of GCK or accelerate product release, which has been observed in some activating GCK mutations. [40] This new interaction site could be exploited for future drug development. In fact, stabilized BAD BH3 domain helices have already shown promising therapeutic utility as they restore proper β-cell function and insulin secretion in BAD-deficient β-cells.…”

“…We recently reported that activating mutations in GCK can result in global conformational alterations. [38,40] It is possible that SUMOylation also alters the conformational dynamics of GCK, as has been observed for other proteins [107,[118][119][120][121], but an investigation into the structural consequences of GCK SUMOylation has yet to be pursued. In particular, it remains to be determined which enzyme conformations of GCK are amenable to SUMOylation.…”

“…Solution NMR, viscosity variation, transient chemical quench-flow kinetics and hydrogen-deuterium exchange mass spectrometry (HDX-MS) were recently used to elucidate the mechanism of action of both variants. [40] These combined efforts demonstrate that unliganded α-activated GCK is characterized by a shift in the conformational ensemble toward a structure that resembles the glucose-bound enzyme. Consequently, α-activation is associated with an alteration in the value of the conformational exchange rate constant, k ex .…”

“…In line with that expectation, two functionally distinct GCK activation mechanisms were recently identified by characterizing genetically engineered GCK mutants: 〈and ®-type activation. [40] Specific mutations in GCK's α13 helix give rise to a variant that symbolizes α-type activation. β-type activation, on the other hand, is exemplified by βhairpin GCK, which contains targeted alterations in the mobile loop region.…”