title: Insights into the biochemical and genetic basis of glucokinase activation from naturally occurring hypoglycemia mutations.
abstract: Glucokinase (GCK) is a key regulatory enzyme in the pancreatic beta-cell and catalyzes the rate-limiting step for beta-cell glucose metabolism. We report two novel GCK mutations (T65I and W99R) that have arisen de novo in two families with familial hypoglycemia. Insulin levels, although inappropriately high for the degree of hypoglycemia, remain regulated by fluctuations in glycemia, and pancreatic histology was normal. These mutations are within the recently identified heterotropic allosteric activator site in the theoretical model of human beta-cell glucokinase. Functional analysis of the purified recombinant glutathionyl S-transferase fusion proteins of T65I and W99R GCK revealed that the kinetic changes result in a relative increased activity index (a measure of the enzyme's phosphorylating potential) of 9.81 and 6.36, respectively, compared with wild-type. The predicted thresholds for glucose-stimulated insulin release using mathematical modeling were 3.1 (T65I) and 2.8 (W99R) mmol/l, which were in line with the patients' fasting glucose. In conclusion, we have identified two novel spontaneous GCK-activating mutations whose clinical phenotype clearly differs from mutations in ATP-sensitive K(+) channel genes. In vitro studies confirm the validity of structural and functional models of GCK and the putative allosteric activator site, which is a potential drug target for the treatment of type 2 diabetes.
authors.affiliation: Department of Diabetes & Vascular Medicine, Peninsula Medical School, Exeter, UK.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Gloyn
authors.forename: Kees
authors.initials: K
authors.lastname: Noordam
authors.forename: Michèl A A P
authors.initials: MA
authors.lastname: Willemsen
authors.forename: Sian
authors.initials: S
authors.lastname: Ellard
authors.forename: Wayne W K
authors.initials: WW
authors.lastname: Lam
authors.forename: Ian W
authors.initials: IW
authors.lastname: Campbell
authors.forename: Paula
authors.initials: P
authors.lastname: Midgley
authors.forename: Chyio
authors.initials: C
authors.lastname: Shiota
authors.forename: Carol
authors.initials: C
authors.lastname: Buettger
authors.forename: Mark A
authors.initials: MA
authors.lastname: Magnuson
authors.forename: Franz M
authors.initials: FM
authors.lastname: Matschinsky
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Hattersley
mesh_terms: D000293:Adolescent; D004252:DNA Mutational Analysis; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007003:Hypoglycemia / Q000201:enzymology* / Q000235:genetics*; D007231:Infant, Newborn; D008297:Male; D008956:Models, Chemical; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D005941:Glucokinase