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  • GK allosteric activators validate the importance of

    2021-11-19

    GK allosteric activators validate the importance of GK from a therapeutic perspective, but the nature of allosteric activation of GK remains unclear. The GK allosteric activator site, the pocket where GKAs bind, is ~20 Å remote from the active site (Fig. 1A). The allosteric site is the locus of many heterozygous GK-activating mutations that are clinically hyperinsulinemic and hypoglycemic [11]. Numerous published structural studies of GK have demonstrated that it undergoes a large conformational change from open to closed upon Losartan binding [3, 12]. However, there is a paucity of structural information on the activating mutants of GK. GKAs and clinically activating mutants were suggested to enhance GK activity via shifting the enzyme structure toward a glucose-bound conformation [3, 12, 13]. In 2008, Christesen et al., found S64Y mutant among four GK activating mutants, in patients who were diagnosed with congenital hyperinsulinemia (CHI). Located in the GK allosteric activator site, the S64Y mutant was biochemically characterized to increase activity in comparison to GK wild-type (GK-wt) [14].
    Materials and methods
    Results and discussion
    Conclusions Since enzyme desolvation is a necessary step during glucose binding, apo-GK-S64Y is more poised than apo-GK-wt to follow the same continuous trajectory of substrate binding. Because apo-GK-S64Y ensemble is more structurally organized, exhibiting limited protein flexibility, and less solvent interactions, apo-GK-S64Y is essentially pre-activated for glucose binding. Lastly, both our glucose binding experiments in the presence of 20% glycerol, as well as in D2O performed by others [40], showed enhancements of glucose binding to GK-wt, indicating that restricted water interactions can facilitate the activation of GK-wt in a manner similar to the de-solvated/pre-activated ground state apo-GK-S64Y. Certainly, the de-solvated/pre-activated ground state of apo-GK-S64Y may provide insights in the design of tunable regulatory elements in protein-based adaptability, and in the development of allosteric activators to treat type 2 diabetes.
    Competing interests
    Acknowledgements We thank the staff at the University of Iowa Protein Crystallography Core, BioCAT beamline 18-ID-D at the Argonne National Laboratory and the SIBYLS beamline 12.3.1 at the Berkeley National Laboratory for assistance with SAXS data collection. We acknowledge the usage of the Advanced Light Source (ALS), which is a DOE Office of Science User Facility under Contract DE-AC02-05CH11231. Additionally, this study used the resources of the Advanced Photon Source (APS), which is supported by the DOE Office of Science and operated by APS under Contract DE-AC02-06CH11357. We thank Dr. Shuxiang Li for assistance with the preliminary MD clustering analysis. This work was supported by the NIH in the forms of Grant R01-GM097373 to M.A.S.
    Introduction Glucose phosphorylation is the initial event in glucose metabolism in all cells and tissue types. In mammalian cells, phosphorylation of glucose in glucose-6-P is catalyzed by hexokinases [1]. The hexokinase IV, commonly called glucokinase or GCK, acts as a glucose sensor in liver and in pancreatic β cells for the maintenance of glucose homeostasis. When glycaemia rises after consumption of carbohydrates GCK reaches its highest activity [2]. As a major regulator of glucose metabolism, GCK deregulation has been associated with development of hyperglycemia in type 2 diabetes. Indeed, Gck mutations either lowering enzyme affinity for glucose or decreasing GCK expression, cause diabetes whereas activating mutations lower blood glucose [3], [4]. As a major actor of glucose homeostasis, GCK is currently considered as a strong candidate target for anti-hyperglycemic drugs for type 2 diabetes [5]. Thus, it is critical to fully understand GCK regulations at both the cellular and molecular levels. Hepatic GCK expression and activity are regulated by both transcriptional and post-transcriptional mechanisms and depend on the fasting and refeeding states of the organism [6]. In contrast to glycolytic genes (l-pyruvate kinase, L-pk) or lipogenic genes (fatty acid synthase, Fas) whose transcription depends on insulin and glucose, Gck transcription is exclusively stimulated by insulin. For example, GCK mRNA and protein levels decrease in liver of insulin-deficient rats and are restored after insulin treatment [7]. Moreover, addition of insulin in primary cultures of rat hepatocytes induces Gck mRNA in a dose-dependent manner [8]. The insulin effect is mediated by the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB or Akt) pathway [9], which triggers SREBP1c binding onto the Gck gene promoter. At the post-transcriptional level, the regulation of hepatic GCK involves the interaction with regulatory proteins like the glucokinase regulatory protein, GKRP. At basal glucose concentrations, around 5 mM, GKRP sequesters GCK in the nucleus in an inactive state. Following high glucose exposure (10–30 mM) or fructose (50 μM–1 mM), GK is released from GKRP and translocates to the cytoplasm [10].