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

Abstract

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 S 0.5 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 Val 62 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 ␤-cells and hepatocytes. Glucokinase (GCK)1 plays a critical role in the regulation of insulin secretion and has been termed the pancreatic ␤-cell glucose sensor on account of its kinetics, which allow the ␤-cells to change glucose phosphorylation rate over a range of physiological glucose concentrations. These kinetic characteristics are the enzyme's low affinity for glucose (S 0.5 ϳ 7.5 mM), cooperativity with glucose (Hill number of ϳ1.7), and lack of inhibition by its product glucose 6-phosphate. Glucokinase plays an important role in glucose sensing not only in the pancreatic ␤-cell but also in the liver and a variety of neural/neuroendocrine cells. These include the pancreatic ␣-cell, L-and K-type gut enterocytes, and certain rare neurons in the central nervous system, mainly in the hypothalamus (1-3). It is the sum of its actions in these multiple sites that ultimately determines the blood glucose concentration. In the liver glucokinase is regulated by glucokinase regulatory protein (GKRP), which acts as a competitive inhibitor with respect to glucose (4, 5). In addition to this role GKRP also determines the subcellular location of glucokinase within the liver cell (6). Glucokinase tra...

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

“…It is possible that this mild thermal lability alone might not be sufficient to account for the hyperglycaemia observed in the patients. Loss of regulation by GCKR and allosteric activators has been reported for other GCK mutants with increased or nearly-normal enzyme activity [18,31]. Despite this, we found that the R397L mutation did not affect GCK-GCKR binding in vitro and was not located in the allosteric site predicted from the crystal structure [30].…”

“…Interestingly, the crystal structure of GCK complexed with a GCK allosteric activator indicates that the M235 residue, together with residues V62, I159, M210, I211 and V452, is involved in hydrophobic interactions with this compound [30]. As expected from this model, MODY2 mutations V62M and M210K result in a loss of effect of GCK activators as well as a loss of inhibition by GCKR [18,31]. It has been proposed that these effects could be responsible for the development of hyperglycaemia.…”

“…The binding of GCKR to GCK is responsible for the inhibition and translocation of the enzyme into the nucleus. Although the physiological importance of GCK regulation by GCKR has been known for sometime, only a few MODY2 mutations have been reported to affect this regulation [18,28,31]. However, and contrary to what might be expected a priori, most of those mutations reduced GCK inhibition by the regulatory protein.…”

“…These results suggest that some of these mutations affect GCK activity through other mechanisms. For example, the involvement of some of these mutations in diabetes has been proposed to be due to an effect on protein stability and/or a loss of regulation by GCKR [17][18][19]. However, the effects of MODY2 mutations on the interaction of GCK with other cellular partners have not been studied previously.…”

Mitochondrial dysfunction in adipocytes: the culprit in type 2 diabetes?

“…This sensor depends on mitochondrial function in addition to a number of other intermediates of the glucose-sensing pathway. Mutations in the other genes of this pathway (including those for glucokinase [GCK], KIR6.2 [KCNJ11] and SUR1 [ABCC8]) present with dysregulated insulin secretion in neonatal life [8,9].…”