Structure Of Glucokinase Research Articles

Clinical characteristics of Algerian subjects with MODY p.R85W glucokinase mutation- in silico assessment of p.R85W effect on glucokinase structure and function

AimMaturity-onset diabetes of the young (MODY) is a genetically and clinically heterogeneous group of autosomal dominantly inherited nonketotic diabetes. MODY2 or (GCK-MODY) subtype is caused by heterozygous mutations in the GCK gene coding for glucokinase. In our current study, we aim to confirm a clinically suspected GCK-MODY phenotype at molecular level and subsequently, to estimate co-segregation of the detected mutation with hyperglycemia among Algerian related subjects. Lastly, assessment of mutation effect was performed using a combination of computational tools. MethodsSixteen related subjects were implicated in this study (proband and 15 relatives). Blood samples were used for biochemical, immunological and genetic assays. The proband was screened for mutation in the GCK gene using PCR-sequencing technique. Mutation screening among proband's relatives was performed by PCR-Restriction fragment length polymorphism (PCR-RFLP) technique. Computational analysis was performed with a combination of predictive tools to assess effect of the detected mutation on glucokinase structure/function and stability. ResultsGenetic analysis results revealed the presence of c.253A>T (p.R85W) GCK mutation at heterozygous state in 11 subjects, while it was absent in 5 others. Biochemical analysis showed that all subjects carrying the p.R85W mutation were affected with diabetes or impaired fasting glucose (fasting glucose 6.94 ± 0.63 mmol/l, HbA1c 6.92 ± 0.77%), whereas the five non-carriers were normoglycemic (fasting glucose 5.01 ± 0.26 mmol/l, HbA1c 5.36 ± 0.17%). All computational tools predict the detected mutation to be ‘damaging’ or ‘probably damaging’ with high scores. ConclusionThe p.R85W GCK mutation segregates with diabetes or IFG in a large pedigree. Clinical features of Algerian GCK-MODY patients are similar to those of other populations; in case of double diabetes the co-occurring diabetes overcomes the GCK-MODY phenotype. In silico predictions results are consistent with metabolic study.

Structure and function of N-acetylglucosamine kinase. Identification of two active site cysteines.

N-Acetylglucosamine is a major component of complex carbohydrates. The mammalian salvage pathway of N-acetylglucosamine recruitment from glycoconjugate degradation or nutritional sources starts with phosphorylation by N-acetylglucosamine kinase. In this study we describe the identification of two active site cysteines of the sugar kinase by site-directed mutagenesis and computer-based structure prediction. Murine N-acetylglucosamine kinase contains six cysteine residues, all of which were mutated to serine residues. The strongest reduction of enzyme activity was found for the mutant C131S, followed by C143S. Determination of the kinetic properties of the cysteine mutants showed that the decreased enzyme activities were due to a strongly decreased affinity to either N-acetylglucosamine for C131S, or ATP for C143S. A secondary structure prediction of N-acetylglucosamine kinase showed a high homology to glucokinase. A model of the three-dimensional structure of N-acetylglucosamine kinase based on the known structure of glucokinase was therefore generated. This model confirmed that both cysteines are located in the active site of N-acetylglucosamine kinase with a potential role in the binding of the transferred gamma-phosphate group of ATP within the catalytic mechanism.