Table 1.
GCK mutations detected in MODY2 children from South Italy.
Table 2.
Kinetic constants of human recombinant wild type-GCK and mutant β-cell GST-GCK fusion proteins.
Figure 1.
View of the mutations p.Phe150Tyr and p.Val154Leu in the whole structures and in their local environment. A
: The structures of GCK in the super-open inactive form (PDB code code: 1v4t) and in the closed active form (PDB code code: 1v4s) are shown on the left and on the right, respectively. In the closed form (right) the sugar is shown as a yellow stick. In both panels, the large domain is in the same orientation and is circled. It is clear that the small domain undergoes a large conformational variation from the super-open to the closed form. Specifically the region embodying the residues Phe150 (blue sphere) and Val154 (red sphere) dramatically changes its orientation. B: Close-up view of the wild-type closed structure showing the hydrophobic core rich in aromatic/hydrophobic amino acids. Leu79, Phe123, Phe148, Phe150, Val154, Leu164, Trp167 and Phe171 form an intricate network of stabilizing hydrophobic interactions. C: Structure of GCK closed structure containing the both the Tyr150 and Leu154 mutated residues. Introduction of the oxydryl group (red) of Tyr150 within the hydrophobic core disrupts the interactions present in the wt-enzyme. The replacement of Val154 by leucine produces only small changes in the closed form.
Figure 2.
Distribution of the selected GCK mutations.
The structure of GCK in the active closed form is shown (PDB code: 1v4s); the small and large domains are drawn in cyan and red, respectively; glucose appears as a yellow stick. Mutation sites are shown as green or blue spheres.
Figure 3.
Effect of temperature on the stability of GST-GCK mutants
. Stock enzyme solutions were diluted to 250 µg/ml in storage buffer containing 30% glycerol, 50 mM glucose, 10 mM glutathione, 5 mM DTT, 200 mM KCl and 50 mMTris/HCl, pH 8.0. Panel A: The enzyme solutions were incubated for 30 min at temperatures ranging from 30 to 55°C and then assayed at 30°C as described in the Methods section. Panel B: The enzyme solutions were incubated for periods of time from 5 to 60 min at 50°C. Results are means and SEM of three independent enzyme preparations for each mutant except for GST-GCK (Phe150Tyr) which corresponds to two independent enzyme preparations. (*) p≤0.03, (†) p<0.008.