• Biochemical characterization of human gluconokinase and the proposed metabolic impact of gluconic acid as determined by constraint based metabolic network analysis.

      Rohatgi, Neha; Nielsen, Tine Kragh; Bjørn, Sara Petersen; Axelsson, Ivar; Paglia, Giuseppe; Voldborg, Bjørn Gunnar; Palsson, Bernhard O; Rolfsson, Óttar; Univ Iceland, Ctr Syst Biol, Reykjavik, Iceland, Univ Iceland, Biomed Ctr, Reykjavik, Iceland, Univ Copenhagen, Fac Hlth Sci, Ctr Prot Res, Copenhagen, Denmark (Public Library Science, 2014)
      The metabolism of gluconate is well characterized in prokaryotes where it is known to be degraded following phosphorylation by gluconokinase. Less is known of gluconate metabolism in humans. Human gluconokinase activity was recently identified proposing questions about the metabolic role of gluconate in humans. Here we report the recombinant expression, purification and biochemical characterization of isoform I of human gluconokinase alongside substrate specificity and kinetic assays of the enzyme catalyzed reaction. The enzyme, shown to be a dimer, had ATP dependent phosphorylation activity and strict specificity towards gluconate out of 122 substrates tested. In order to evaluate the metabolic impact of gluconate in humans we modeled gluconate metabolism using steady state metabolic network analysis. The results indicate that significant metabolic flux changes in anabolic pathways linked to the hexose monophosphate shunt (HMS) are induced through a small increase in gluconate concentration. We argue that the enzyme takes part in a context specific carbon flux route into the HMS that, in humans, remains incompletely explored. Apart from the biochemical description of human gluconokinase, the results highlight that little is known of the mechanism of gluconate metabolism in humans despite its widespread use in medicine and consumer products.
    • Kinetic analysis of gluconate phosphorylation by human gluconokinase using isothermal titration calorimetry.

      Rohatgi, Neha; Guðmundsson, Steinn; Rolfsson, Óttar; Univ Iceland, Ctr Syst Biol, IS-101 Reykjavik, Iceland, Univ Iceland, Biomed Ctr, IS-101 Reykjavik, Iceland (Elsevier Science BV, 2015-11-30)
      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.
    • Mass conserved elementary kinetics is sufficient for the existence of a non-equilibrium steady state concentration.

      Fleming, R M T; Thiele, I; [ 1 ] Univ Iceland, Ctr Syst Biol, IS-101 Reykjavik, Iceland [ 2 ] Univ Iceland, Fac Med, Dept Biochem & Mol Biol, IS-101 Reykjavik, Iceland [ 3 ] Univ Iceland, Fac Ind Engn Mech Engn & Comp Sci, IS-101 Reykjavik, Iceland (Academic Press Ltd- Elsevier Science, 2012-12-07)
      Living systems are forced away from thermodynamic equilibrium by exchange of mass and energy with their environment. In order to model a biochemical reaction network in a non-equilibrium state one requires a mathematical formulation to mimic this forcing. We provide a general formulation to force an arbitrary large kinetic model in a manner that is still consistent with the existence of a non-equilibrium steady state. We can guarantee the existence of a non-equilibrium steady state assuming only two conditions; that every reaction is mass balanced and that continuous kinetic reaction rate laws never lead to a negative molecule concentration. These conditions can be verified in polynomial time and are flexible enough to permit one to force a system away from equilibrium. With expository biochemical examples we show how reversible, mass balanced perpetual reaction(s), with thermodynamically infeasible kinetic parameters, can be used to perpetually force various kinetic models in a manner consistent with the existence of a steady state. Easily testable existence conditions are foundational for efforts to reliably compute non-equilibrium steady states in genome-scale biochemical kinetic models.