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 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.
- SourceAvailable from: Dr. Nandakumar Yellapu[Show abstract] [Hide abstract]
ABSTRACT: Glucokinase (GK) plays a critical role in glucose homeostasis and the mutations in GK gene results in pathogenic complications known as Maturity Onset Diabetes of the Young2 (MODY2), an autosomal dominant form of diabetic condition. In the present study GK was purified from human liver tissue and the pure enzyme showed single band in SDS-PAGE with a molecular weight of 50 KD. The kinetics of pure GK showed enzyme activity of 0.423 ± 0.02 μM glucose-6-phosphate/ml/min and Km value of 6.66 ± 0.02 μM. These values were compared in the liver biopsy of clinically proven type2 diabetic patient, where GK kinetics showed decreased enzyme activity of 0.16 ± 0.025 μM glucose-6-phosphate/ml/min and increased Km of 23 ± 0.9 μM indicating the hyperglycemic condition in the patient. The genetic analysis of 10(th) exon of GK gene from this patient showed R308K mutation. In order to substantiate these results, a comparative molecular dynamics and docking studies were carried out where higher docking score (-10.218 Kcal/mol) was observed in the mutated GK than wild type GK structure (-12.593 Kcal/mol) indicating affinity variations for glucose. During the simulation process glucose was expelled out from the mutant conformation but not form wild type GK making glucose unavailable for phosphorylation. Therefore, these results conclusively explain hyperglycemic condition in this patient. This article is protected by copyright. All rights reserved.Biotechnology and Applied Biochemistry 01/2014; · 1.35 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Background Chronic kidney disease (CKD) is well known as a strong risk factor for both of end-stage renal disease and cardiovascular disease. To clarify the association of glucokinase and glucokinase regulatory protein (GCKR) polymorphisms with the risk of CKD in Japan, we examined this association among Japanese individuals using cross-sectional data. Methods The subjects for this analysis were 3,314 consecutively selected participants from the Japan Multi-Institutional Collaborative Cohort Study. Age- and sex- adjusted odds ratios (aORs) of CKD stages 3–5 were calculated for each genotype by logistic regression and the effects of genotype on estimated glomerular filtration rate were evaluated by linear regression. Gene–environment interaction was also investigated based on questionnaire information. Results When subjects with GCKR rs780094 G/A and G/G, or GCKR rs1260326 T/C and C/C were combined together and compared with the references (GCKR rs780094 A/A or GCKR rs1260326 T/T), the aORs were 0.84 (0.69–1.02) or 0.81 (0.67–0.99) (p = 0.075 or 0.037), respectively. A significant OR for interaction between GCKR rs1260326 T/T and current smoking (OR = 1.79, p = 0.041) was also observed. Conclusion The present study suggests a possible association of the T/T genotype of GCKR rs1260326 polymorphism with elevated risk of CKD and its interaction with current smoking, which may support the possibility of performing risk evaluation and prevention of this potentially life-threatening disease based on genetic traits in the near future.Journal of nephrology 01/2013; · 2.02 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Diabetes mellitus is a chronic metabolic disorder involving the dysregulation of glucose metabolism, β-cell dysfunction, and impaired insulin sensitivity. Glucokinase (GK) promotes glycogen synthesis, while it enhances insulin secretion from pancreatic β-cells. In this study, we focused on molecular modeling study of 3-alkoxy-5-phenoxy-N-thiazolyl benzamide analogs with reference to structural requirements. The amalgamated best fit consensus scoring function showed coefficient of determination (0.927), leave-one-out cross-validated squared correlation coefficient (0.865), and external predictivity value (0.763). The binding of 3-alkoxy-5-phenoxy-N-thiazolyl benzamide analogs to glucokinase enzyme was explored with the help of docking. The most stable ligand–enzyme complex of compound TR-2 showed that the NH of the benzamide make key hydrogen bonds with the backbone C=O of Arg63. The phenoxy moiety on the 5th position of benzene ring occupies the hydrophobic space on the allosteric binding site constituted from Met210, Met235, Cys220, and Tyr214. One of the oxygen of methylsulfonyl group forms hydrogen bond with NE2 of Gln98 and phenyl ring and the aromatic ring of Tyr215 are perpendicular to each other, which probably increase potency due to van der Waals interactions.The structural insights gleaned from the study could be usefully employed to design activators with a much more enhanced potency.Medicinal Chemistry Research 01/2012; · 1.61 Impact Factor