Effects of novel maturity-onset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability

摘要: Glucokinase acts as the pancreatic glucose sensor and plays a critical role in regulation of insulin secretion by β-cell. Heterozygous mutations glucokinase-encoding GCK gene, which result reduction enzymatic activity, cause monogenic form diabetes, MODY2 (maturity-onset diabetes young 2). We have identified functionally characterized missense gene diabetic families that protein Leu165→Phe, Glu265→Lys Thr206→Met. The first two are novel co-segregate with phenotype their respective not found more than 50 healthy control individuals. In order to measure biochemical effects these on glucokinase we bacterially expressed affinity-purified islet human proteins carrying fused GST (glutathione S-transferase). Enzymatic assays recombinant revealed Thr206→Met Leu165→Phe strongly affect kinetic parameters glucokinase, agreement localization both residues close active site enzyme. contrast, mutation Glu265→Lys, has weaker effect kinetics affects stability, suggesting possible structural defect this mutant protein. Finally, none tested appears interaction gluco-kinase regulatory yeast two-hybrid system.