Insights into the Structure and Regulation of Glucokinase from a Novel Mutation (V62M), Which Causes Maturity-onset Diabetes of the Young

The Children's Hospital of Philadelphia, Filadelfia, Pennsylvania, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2005; 280(14):14105-13. DOI: 10.1074/jbc.M413146200
Source: PubMed


Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.

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    • "However, unlike other hexokinases, it is not inhibited by its product glucose-6-phosphate and maintains high glycolytic flux in the presence of elevated glucose, coupling carbohydrate sensing to insulin secretion in the b-cell (Bedoya et al. 1986, Alvarez et al. 2002, Newsholme & Krause 2012). Alterations in the activity of important glycolytic enzymes such as GCK and phosphofructokinase can modulate GSIS, and this may lead to impaired glucose metabolism and insulin secretion (Nielsen et al. 1998, Westermark & Lansner 2003, Gloyn et al. 2005). Furthermore, chronic hyperglycaemic conditions, as observed in T2DM, can negatively regulate the expression of several important glucose metabolising b-cell genes including SLC2A2, GCK, Ca 2C channels and insulin transcription factors pancreatic and duodenal homoeobox 1 (Pdx1), neurogenic differentiation 1 (NeuroD1) and v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafA) (Cnop et al. 2005, Newsholme et al. 2010). "
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    • "The combination of these two features indicates that GCK can maintain glycolytic flux in the face of high-glucose load (Fu et al., 2013). Interestingly, it has been suggested that changes in GCK function resulted in decreased GSIS that could possibly lead to DM, thus highlighting the importance of this metabolic step (Gloyn et al., 2005; Rorsman & Braun, 2013). In addition, another glycolytic enzyme, phosphofructokinase (PFK), is also an important regulatory site in glycolysis and is allosterically controlled by ATP levels "
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    • "In these instances other mutational mechanisms may be at play, for instance, mechanisms that effect enzyme stability [Burke et al., 1999] or binding with regulatory molecules such at GKRP [Arden et al., 2007; Gloyn et al., 2005] or the bifunctional enzyme PFK-2/FBPase-2 [Arden et al., 2007] or perhaps with an unknown endogenous allosteric activator [Gloyn et al., 2005]. For example, the V62M GCK- MODY mutation appears be paradoxically kinetically activating as opposed to inactivating [Gloyn et al., 2005], and has been shown to have a lack of inhibition by liver GKRP and a lack of activation by pharmacological GCK small molecular activators [Gloyn et al., 2005], although studies in min6 cells have suggested that the V62M GCK mutation may result in catalytic instability [Arden et al., 2007]. There is evidence that at least nine other GCK- MODY mutations affect enzyme stability from recombinant mutant GCK protein studies [Burke et al., 1999; Davis et al., 1999; Galan et al., 2006; Garcia-Herrero et al., 2007; Gloyn et al., 2005; Kesavan et al., 1997; Sagen et al., 2006]. "
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