Thermodynamics of reactions catalyzed by anthranilate synthase

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthrani late synthase, the enzyme located at the first step in the biosynthetic pat hway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(a q) = anthranilate(aq) + pyruvate(aq) + H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisoch orismate (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. Microcalor imetric measurements were performed on the overall reaction at a temperatur e of 298.15 K and pH 7.79. Equilibrium measurements were performed on the f irst partial (ADIC synthase) reaction at temperatures ranging from 288.15 t o 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibr ium and calorimetric measurements were analyzed in terms of a chemical equi librium model that accounts for the multiplicity of ionic states of the rea ctants and products. These calculations gave thermodynamic quantities at th e temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate(2-) (aq) + NH4+(aq) = anthranilate(-)(aq) + pyruvate(-)(aq) + H+(aq) + H2O(l), Delta(r)H(m)degrees = -(116.3 +/- 5.4) kJ mol(-1). For the reaction: choris mate(2-)(aq) + NH4+(aq) = ADIC(-)(aq) + H2O(l), K = (20.3 +/- 4.5) and Delt a(r)H(m)degrees = (7.5 +/- 0.6) kJ mol(-1). Thermodynamic cycle calculation s were used to calculate thermodynamic quantities for three additional reac tions that are pertinent to this branch point of the chorismate pathway. Th e quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ion ic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes Delta(r)G(m)'degrees under approximately p hysiological conditions are given. (C) 2000 Elsevier Science B.V. All right s reserved.