title: Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors.
abstract: Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-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 (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 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.
authors.affiliation: Sanofi Aventis Deutschland GmbH, Research and Development, D-65926 Frankfurt am Main, Germany. oliver.anderka@sanofi-aventis.com
authors.forename: Oliver
authors.initials: O
authors.lastname: Anderka
authors.forename: Janina
authors.initials: J
authors.lastname: Boyken
authors.forename: Ursula
authors.initials: U
authors.lastname: Aschenbach
authors.forename: Andreas
authors.initials: A
authors.lastname: Batzer
authors.forename: Oliver
authors.initials: O
authors.lastname: Boscheinen
authors.forename: Dieter
authors.initials: D
authors.lastname: Schmoll
mesh_terms: D000818:Animals; D001703:Biophysics; D002021:Buffers; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005941:Glucokinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013696:Temperature; D013816:Thermodynamics
chemical_list: D002021:Buffers; D002352:Carrier Proteins; D011994:Recombinant Proteins; C080906:glucokinase regulatory protein; D005941:Glucokinase