Glucokinase acts as the pancreatic glucose sensor and plays a critical role in the regulation of insulin secretion by the β-cell. Heterozygous mutations in the glucokinase-encoding GCK gene, which result in a reduction of the enzymatic activity, cause the monogenic form of diabetes, MODY2 (maturity-onset diabetes of the young 2). We have identified and functionally characterized missense mutations in the GCK gene in diabetic families that result in protein mutations Leu165→Phe, Glu265→Lys and Thr206→Met. The first two are novel GCK mutations that co-segregate with the diabetes phenotype in their respective families and are not found in more than 50 healthy control individuals. In order to measure the biochemical effects of these missense mutations on glucokinase activity, we bacterially expressed and affinity-purified islet human glucokinase proteins carrying the respective mutations and fused to GST (glutathione S-transferase). Enzymatic assays on the recombinant proteins revealed that mutations Thr206→Met and Leu165→Phe strongly affect the kinetic parameters of glucokinase, in agreement with the localization of both residues close to the active site of the enzyme. In contrast, mutation Glu265→Lys, which has a weaker effect on the kinetics of glucokinase, strongly affects the protein stability, suggesting a possible structural defect of this mutant protein. Finally, none of the mutations tested appears to affect the interaction of gluco-kinase with the glucokinase regulatory protein in the yeast two-hybrid system.
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Research Article|
December 12 2005
Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability
María Galán;
María Galán
*Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid 28040, Spain
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Olivier Vincent;
Olivier Vincent
†Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas del CSIC, Madrid 28040, Spain
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Isabel Roncero;
Isabel Roncero
*Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid 28040, Spain
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Sharona Azriel;
Sharona Azriel
‡Servicio de Endocrinología, Hospital 12 de Octubre, Av. de Córdoba s/n, Madrid 28041, Spain
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Pedro Boix-Pallares;
Pedro Boix-Pallares
§Servicio de Endocrinología, Hospital General de Asturias, Julián Clavería s/n, Oviedo 33006, Spain
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Elías Delgado-Alvarez;
Elías Delgado-Alvarez
§Servicio de Endocrinología, Hospital General de Asturias, Julián Clavería s/n, Oviedo 33006, Spain
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Francisco Díaz-Cadórniga;
Francisco Díaz-Cadórniga
§Servicio de Endocrinología, Hospital General de Asturias, Julián Clavería s/n, Oviedo 33006, Spain
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Enrique Blázquez;
Enrique Blázquez
*Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid 28040, Spain
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María-Angeles Navas
María-Angeles Navas
1
*Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense de Madrid, Ciudad Universitaria, Madrid 28040, Spain
1To whom correspondence should be addressed (email manavas@med.ucm.es).
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Publisher: Portland Press Ltd
Received:
July 15 2005
Revision Received:
August 26 2005
Accepted:
September 21 2005
Accepted Manuscript online:
September 21 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 393 (1): 389–396.
Article history
Received:
July 15 2005
Revision Received:
August 26 2005
Accepted:
September 21 2005
Accepted Manuscript online:
September 21 2005
Citation
María Galán, Olivier Vincent, Isabel Roncero, Sharona Azriel, Pedro Boix-Pallares, Elías Delgado-Alvarez, Francisco Díaz-Cadórniga, Enrique Blázquez, María-Angeles Navas; Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability. Biochem J 1 January 2006; 393 (1): 389–396. doi: https://doi.org/10.1042/BJ20051137
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