Kinetic analysis of gluconate phosphorylation by human gluconokinase using isothermal titration calorimetry.
Gluconate is a commonly encountered nutrient, which is degraded by the enzyme gluconokinase to generate 6-phosphogluconate. Here we used isothermal titration calorimetry to study the properties of this reaction. ΔH, KM and kcat are reported along with substrate binding data. We propose that the reaction follows a ternary complex mechanism, with ATP binding first. The reaction is inhibited by gluconate, as it binds to an Enzyme-ADP complex forming a dead-end complex. The study exemplifies that ITC can be used to determine mechanisms of enzyme catalyzed reactions, for which it is currently not commonly applied.
Center for Systems Biology, University of Iceland, Sturlugata 8, 101 Reykjavik, Iceland; University of Iceland Biomedical Center, Laeknagardur, 101 Reykjavik, Iceland.
Neha
N
Rohatgi
Center for Systems Biology, University of Iceland, Sturlugata 8, 101 Reykjavik, Iceland.
Steinn
S
Guðmundsson
Center for Systems Biology, University of Iceland, Sturlugata 8, 101 Reykjavik, Iceland; University of Iceland Biomedical Center, Laeknagardur, 101 Reykjavik, Iceland. Electronic address: ottarr@hi.is.
Óttar
Ó
Rolfsson
D000244:Adenosine Diphosphate / Q000378:metabolism; D002151:Calorimetry; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005942:Gluconates / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation / Q000187:drug effects; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013696:Temperature
D004791:Enzyme Inhibitors; D005942:Gluconates; D000244:Adenosine Diphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C020578:gluconokinase; C030691:gluconic acid
Enzyme kinetics; Gluconate (Glcn); Human gluconokinase (hGntK, IdnK); Human metabolism; Isothermal titration calorimetry