title: Glucokinase thermolability and hepatic regulatory protein binding are essential factors for predicting the blood glucose phenotype of missense mutations.
abstract: To better understand how glucokinase (GK) missense mutations associated with human glycemic diseases perturb glucose homeostasis, we generated and characterized mice with either an activating (A456V) or inactivating (K414E) mutation in the gk gene. Animals with these mutations exhibited alterations in their blood glucose concentration that were inversely related to the relative activity index of GK. Moreover, the threshold for glucose-stimulated insulin secretion from islets with either the activating or inactivating mutation were left- or right-shifted, respectively. However, we were surprised to find that mice with the activating mutation had markedly reduced amounts of hepatic GK activity. Further studies of bacterially expressed mutant enzymes revealed that GK(A456V) is as stable as the wild type enzyme, whereas GK(K414E) is thermolabile. However, the ability of GK regulatory protein to inhibit GK(A456V) was found to be less than that of the wild type enzyme, a finding consistent with impaired hepatic nuclear localization. Taken together, this study indicates that it is necessary to have knowledge of both thermolability and the interactions of mutant GK enzymes with GK regulatory protein when attempting to predict in vivo glycemic phenotypes based on the measurement of enzyme kinetics.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Maria F
authors.initials: MF
authors.lastname: Pino
authors.forename: Kyoung-Ah
authors.initials: KA
authors.lastname: Kim
authors.forename: Kathy D
authors.initials: KD
authors.lastname: Shelton
authors.forename: Jill
authors.initials: J
authors.lastname: Lindner
authors.forename: Stella
authors.initials: S
authors.lastname: Odili
authors.forename: Changhong
authors.initials: C
authors.lastname: Li
authors.forename: Heather W
authors.initials: HW
authors.lastname: Collins
authors.forename: Masakazu
authors.initials: M
authors.lastname: Shiota
authors.forename: Franz M
authors.initials: FM
authors.lastname: Matschinsky
authors.forename: Mark A
authors.initials: MA
authors.lastname: Magnuson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D001786:Blood Glucose / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044882:Glucose Metabolism Disorders / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D006358:Hot Temperature; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D020125:Mutation, Missense; D010641:Phenotype; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001786:Blood Glucose; D002352:Carrier Proteins; C499483:Gckr protein, mouse; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D005941:Glucokinase