Thermodynamics of reactions catalyzed by anthranilate synthase Original Research Article

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq)+ammonia(aq)=anthranilate(aq)+pyruvate(aq)+H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq)+ammonia(aq)=ADIC(aq)+H2O(l) and ADIC(aq)=anthranilate(aq)+pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate2−(aq)+NH4+(aq)=anthranilate−(aq)+pyruvate−(aq)+H+(aq)+H2O(l), ΔrHmo=−(116.3±5.4) kJ mol−1. For the reaction: chorismate2−(aq)+NH4+(aq)=ADIC−(aq)+H2O(l), K=(20.3±4.5) and ΔrHmo=(7.5±0.6) kJ mol−1. Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes ΔrG′mo under approximately physiological conditions are given.