Biophysical Characterization of the Interaction between Hepatic Glucokinase and Its Regulatory Protein

Abstract

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic ␤-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (K d ‫؍‬ 45 nM, ⌬H ‫؍‬ 15.6 kcal/mol, T⌬S ‫؍‬ 25.7 kcal/mol, ⌬C p ‫؍‬ ؊354 cal mol ؊1 K ؊1 ). With k off ‫؍‬ 1.3 ؋ 10 ؊2 s ؊1 , the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs. Glucokinase (GK)2 is the predominant glucose-phosphorylating enzyme in liver and pancreatic ␤-cells and plays a central role in blood glucose homeostasis (1, 2). Enhancing GK activity by small molecule GK activators (GKAs) is currently under evaluation as an approach for the treatment of diabetes (3). Hepatic GK activity is controlled by an endogenous inhibitor, a 68-kDa GK regulatory protein (GKRP) (4). During starvation, the enzyme is bound to GKRP, leading to its inactivation and sequestration in the nucleus. After refeeding, the GK-GKRP complex dissociates and GK translocates into the cytoplasm (5, 6). In a rodent model of type 2 diabetes, this translocation is impaired, which could contribute to the defective blood glucose homeostasis (7). Further evidence for the metabolic impact of GKRP comes from recent human genome-wide association studies that show a strong linkage between a GKRP gene polymorphism and serum triglyceride levels (8, 9). The formation of the GK-GKRP complex is favored by fructose-6-phosphate (F6P) and inhibited by fructose-1-phosphate (F1P) (10). Both sugar phosphates bind to the same site on the regulatory protein (11). An increase of the intracellular F1P concentration leading to the release of GK from its inhibitory complex with GKRP is most likely the reason for the stimulation of hepatic glucose metabolism by catalytic amounts of fructose or sorbitol (6, 12, 13). Site-directed mutagenesis experiments indicate that the binding interface for GKRP lies close to the binding site of allosteric GKAs of the amino-heteroaryl-amide type (14). However, it is controversial if thes...

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