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

Abstract

Glucokinase acts as the pancreatic glucose sensor and plays a critical role in the regulation of insulin secretion by the beta-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.

Figure 1. Localization of the mutated residues Leu¹⁶⁵, Thr²⁰⁶ and Glu²⁶⁵ on the structural model for the β-cell GK and multiple sequences alignment of GKs and hexokinases from different species at the positions where mutations were identified

(A) Closed conformation (PDB code 1V4S) of wild-type GK [14] is represented using the RasMol program [48]. Mutated residues in the MODY families are indicated as black sticks and ribbons. The position of glucose is indicated as a black sphere. (B) Alignment of human (Hum) GK (GenBank® accession number NP_277042), mouse liver GK (GenBank® accession number NP_034422), rat liver GK (GenBank® accession number NP_036697), Xenopus (Xen) liver GK (GenBank® accession number X93494), rat hexokinase I (GenBank® accession number NP_036866) and rat hexokinase II (GenBank® accession number NP_036867) using the ClustalW program. Numbers indicate amino acid positions in the proteins. Boxes indicate the conservation of the corresponding residues.

Figure 2. Glucose-dependent activity of the mutated forms of GK

Specific activity at each glucose concentration was calculated as described in the Experimental section. Results are means±S.E.M. for n separate enzyme expressions.

Figure 3. Two-hybrid interaction between GK and mutant derivatives and the GKRP

Two-hybrid interaction of Gal4-binding domain–GRKP with Gal4-activating domain–GK and its mutant derivatives were tested in S. cerevisiae strain Y187. Filter lift assays were developed for 1 h. In each case, three independent transformants were tested, with similar results.

Figure 4. Effects of temperature on the stability of GST–GK variants

Stock enzyme solutions were diluted to 250 μg/ml in storage buffer containing 30% (v/v) glycerol, 50 mM glucose, 10 mM glutathione, 5 mM DTT, 200 mM KCl and 50 mM Tris/HCl, pH 8.0. (A) The enzyme solutions were incubated for 30 min at different temperatures ranging from 30 to 55 °C and then assayed at 30 °C as described in the Experimental section. (B) The enzyme solutions were incubated for different periods of time from 5 to 60 min at 50 °C. Means±S.E.M. for three independent enzyme preparations are shown for each case.