Tewari Yadu B, Jensen Pia Y, Kishore Nand, Mayhew Martin P, Parsons James F, Eisenstein Edward, Goldberg Robert N
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Biophys Chem. 2002 Apr 10;96(1):33-51. doi: 10.1016/s0301-4622(02)00034-0.
Microcalorimetry and high-performance liquid chromatography (HPLC) have been used to conduct a thermodynamic investigation of reactions catalyzed by PABA synthase, the enzyme located at the first step in the shikimic acid metabolic pathway leading from chorismate to 4-aminobenzoate (PABA). The overall biochemical reaction catalyzed by the PabB and PabC components of PABA synthase is: chorismate(aq)+ammonia(aq)=4-aminobenzoate(aq)+pyruvate(aq)+H(2)O(l). This reaction can be divided into two partial reactions involving the intermediate 4-amino-4-deoxychorismate (ADC): chorismate(aq)+ammonia(aq)=ADC(aq)+H(2)O(l) and ADC(aq)=4-aminobenzoate(aq)+pyruvate(aq). Microcalorimetric measurements were performed on all three of these reactions at a temperature of 298.15 K and pH values in the range 8.72-8.77. Equilibrium measurements were performed on the first partial (ADC synthase) reaction at T=298.15 K and at pH=8.78. The saturation molality of 4-aminobenzoate(cr) in water is (0.00382+/-0.0004) mol kg(-1) at T=298.15 K. 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: chorismate(2-)(aq)+NH(4)(+)(aq)=ADC(-)(aq)+H(2)O(l), K=(10.8+/-4.2) and Delta(r)H(m)(o)=-(35+/-15) kJ mol(-1). For the reaction: ADC(-)(aq)=4-aminobenzoate(-)(aq)+pyruvate(-)(aq)+H(+)(aq), Delta(r)H(m)(o)=-(139+/-23) kJ mol(-1). For the reaction: chorismate(2-)(aq)+NH(4)(+)(aq)=4-aminobenzoate(-)(aq)+pyruvate(-)(aq)+H(2)O(l)+H(+)(aq), Delta(r)H(m)(o)=-(174+/-6) kJ mol(-1). Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that utilize L-glutamine rather than ammonia and that are pertinent to this branch point of the shikimic acid 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 Delta(r)G'(m)(o) under approximately physiological conditions are given.
微量热法和高效液相色谱法（HPLC）已被用于对由对氨基苯甲酸合酶催化的反应进行热力学研究，该酶位于莽草酸代谢途径从分支酸到4-氨基苯甲酸（PABA）的第一步。由对氨基苯甲酸合酶的PabB和PabC组分催化的整体生化反应为：分支酸（水溶液）+氨（水溶液）=4-氨基苯甲酸（水溶液）+丙酮酸（水溶液）+H₂O（液态）。该反应可分为两个涉及中间体4-氨基-4-脱氧分支酸（ADC）的部分反应：分支酸（水溶液）+氨（水溶液）=ADC（水溶液）+H₂O（液态）和ADC（水溶液）=4-氨基苯甲酸（水溶液）+丙酮酸（水溶液）。在298.15K的温度和8.72 - 8.77范围内的pH值下，对这三个反应都进行了微量热测量。在T = 298.15K和pH = 8.78时，对第一个部分（ADC合酶）反应进行了平衡测量。在T = 298.15K时，4-氨基苯甲酸（cr）在水中的饱和质量摩尔浓度为(0.00382±0.0004) mol kg⁻¹。根据一个考虑反应物和产物离子态多重性的化学平衡模型，对平衡和量热测量结果进行了分析。这些计算给出了在298.15K温度和离子强度为零的情况下，涉及特定离子形式的化学参考反应的热力学量。对于反应：分支酸²⁻（水溶液）+NH₄⁺（水溶液）=ADC⁻（水溶液）+H₂O（液态），K=(10.8±4.2)且ΔrHm⁰=-(35±15) kJ mol⁻¹。对于反应：ADC⁻（水溶液）=4-氨基苯甲酸⁻（水溶液）+丙酮酸⁻（水溶液）+H⁺（水溶液），ΔrHm⁰=-(139±23) kJ mol⁻¹。对于反应：分支酸²⁻（水溶液）+NH₄⁺（水溶液）=4-氨基苯甲酸⁻（水溶液）+丙酮酸⁻（水溶液）+H₂O（液态）+H⁺（水溶液），ΔrHm⁰=-(174±6) kJ mol⁻¹。使用热力学循环计算来计算另外三个利用L-谷氨酰胺而非氨且与莽草酸途径这个分支点相关的反应的热力学量。本研究中获得的量允许计算这些反应的平衡位置作为温度、pH和离子强度的函数。给出了在近似生理条件下的表观平衡常数和标准转化吉布斯自由能变化ΔrG'm⁰的值。
