Ž . Biophysical Chemistry 84 2000 4564 Thermodynamics of reactions catalyzed by anthranilate synthase W. Malcolm Byrnes, Robert N. Goldberg , Marcia J. Holden, Martin P. Mayhew, Yadu B. Tewari Biotechnology Di ision, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Received 1 November 1999; received in revised form 23 November 1999; accepted 23 November 1999 Abstract 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 Ž . Ž . Ž . Ž . Ž. synthase is: chorismate aq ammonia aq anthranilate aq pyruvate aq H O l . This reaction can be divided 2 Ž . Ž . into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate ADIC : chorismate aq Ž . Ž . Ž. Ž . Ž . Ž . ammonia aq ADIC aq HOl and ADIC aq anthranilate aq pyruvate aq . The native anthranilate syn- 2 thase 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. Ž . 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: 2 Ž . Ž . Ž . Ž . Ž . Ž. o Ž . chorismate aq NH aq anthranilate aq pyruvate aq H aq HOl, H 116.3 5.4 kJ 4 2 r m 1 2 Ž . Ž . Ž . Ž. Ž . o Ž mol . For the reaction: chorismate aq NH aq ADIC aq HOl, K 20.3 4.5 and H 7.5 4 2 r m . 1 0.6 kJ mol . 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 Corresponding author. Building 227, Room A215, 100 Bureau Drive, Mail Stop 8312, National Institute of Standards and Technology, Gaithersburg, MD 20899-8312, USA. Tel.: 1-301-975-2584; fax: 1-301-975-5449 or 1-301-330-3447. Ž . E-mail address: robert.goldberg@nist.gov R.N. Goldberg 0301-462200$ - see front matter 2000 Elsevier Science B.V. All rights reserved. Ž . PII: S 0 3 0 1 - 4 6 2 2 99 00145-3