Abstract
Aims/hypothesisThe mammalian enzyme glucokinase (GK), expressed predominantly in liver and pancreas, plays an essential role in carbohydrate metabolism. Monogenic GK disorders emphasise the role of GK in determining the blood glucose set point.MethodsA family with congenital hyperinsulinism (CHI) was examined for GCK gene variants by Sanger sequencing. A combined approach, involving kinetic analysis (also using GK activators and inhibitors), intracellular translocation assays, insulin secretion measurements and structural modelling, was used to investigate the novel variant compared with known variants.ResultsWe report on the novel gain-of-function GCK variant p.Val455Leu (V455L), inherited as an autosomal dominant trait in a German family with CHI and concomitant obesity (fasting blood glucose 2.1 mmol/l, BMI 45.0 kg/m2, HOMA-IR 1.5 in an adult female family member); one male family member developed type 2 diabetes until age 35 years (with fasting glucose 2.8–3.7 mmol/l, BMI 38.9 kg/m2, HOMA-IR 4.6). Kinetic characterisation of the V455L variant revealed a significant increase in glucose affinity (glucose concentration at which reaction rate is half its maximum rate [S0.5]: mutant 2.4 ± 0.3 mmol/l vs wild-type 7.6 ± 1.0 mmol/l), accompanied by a distinct additive susceptibility to both the endogenous activator fructose 2,6-bisphosphatase and the synthetic allosteric activator RO-28-1675. The effect of RO-28-1675 was more pronounced when compared with the previously known GK variants V455M and V455E. Binding to the inhibitor glucokinase regulatory protein was unimpaired for V455L and V455E but was reduced for V455M, whereas mannoheptulose inhibited all GK variants and the wild-type enzyme. Structural analyses suggested a role for residue 455 in rearrangements between the inactive and active conformations of GK and also in allosteric activation. Comparison with V455M and V455E and an overview of activating GK variants provided a context for the novel sequence aberration in terms of altered GK enzyme characteristics caused by single amino acid changes.Conclusion/interpretationWe provide new knowledge on the structure–function relationship of GK, with special emphasis on enzyme activation, potentially yielding fresh strategic insights into breaking the vicious circle of fluctuating blood glucose levels and the attendant risk of long-lasting metabolic changes in both CHI and type 2 diabetes.Graphical abstract
Highlights
The mammalian glucose-phosphorylating enzyme glucokinase (GK; hexokinase IV) plays a central role in glucose homeostasis
Activity-diminishing GK variants cause MODY2 or permanent neonatal diabetes mellitus (PNDM), whereas activating GK variants result in congenital hyperinsulinism (CHI)
We systematically evaluate the kinetic properties of the novel variant V455L in comparison with those of the wild-type enzyme and previously described naturally occurring CHI and MODY2 mutants, respectively, V455M [12, 13] and V455E [14, 15]
Summary
Novel activating GCK missense variant p.Val455Leu In the course of medical examinations conducted in a German family in order to elucidate the cause of persistent hyperinsulinaemic hypoglycaemia of infancy, sequencing of the GCK gene identified heterozygosity for the novel missense variant c.1363G>C, predicting an amino acid replacement p.Val455Leu. While the inherently low activity of the MODY2 mutant GK-E455 was enhanced by FBPase-2 in a proportion similar to the wild-type enzyme, this variant was only slightly activated by RO-28-1675 For both activating GK mutants (at 2 mmol/l glucose for GKL455 half maximal effective concentration [EC50] = 3.97 ± 1.49 μmol/l and for GK-M455 EC50 = 7.19 ± 2.65 μmol/l), RO-28-1675 activation was less pronounced than for the wild-type enzyme (at 2 mmol/l glucose EC50 = 0.88 ± 0.29 μmol/l), as reported previously for treatment of GKM455 with a closely related GKA [33]. In the super-open, inactive conformation of GK, the wild-type residue V455 makes hydrophobic contacts with side chains of a β-sheet cluster within the small domain of the enzyme, V89 and V101 of sheets β3 and β4 (Fig. 5a,b). The leucine side chain at position 455 will not impede binding of RO-28-1675, and even stabilisation of the complex due to hydrophobic interactions with either I211 (α5) or V62 (interconnecting loop) seems plausible (Fig. 7e)
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