Department of Chemistry, Hugel Science Center, Lafayette College, Easton, Pennsylvania 18042, USA.
J Phys Chem B. 2011 Nov 24;115(46):13746-54. doi: 10.1021/jp208129a. Epub 2011 Nov 3.
Binding of a cis,syn-cyclobutane pyrimidine dimer (CPD) to Escherichia coli DNA photolyase was examined as a function of temperature, enzyme oxidation state, salt, and substrate conformation using isothermal titration calorimetry. While the overall ΔG° of binding was relatively insensitive to most of the conditions examined, the enthalpic and entropic terms that make up the free energy of binding are sensitive to the conditions of the experiment. Substrate binding to DNA photolyase is generally driven by a negative change in enthalpy. Electrostatic interactions and protonation are affected by the oxidation state of the required FAD cofactor and substrate conformation. The fully reduced enzyme appears to bind approximately two additional water molecules as part of substrate binding. More significantly, the experimental change in heat capacity strongly suggests that the CPD lesion must be flipped out of the intrahelical base stacking prior to binding to the protein; the DNA repair enzyme appears to recognize a solvent-exposed CPD as part of its damage recognition mechanism.
采用等温滴定量热法，研究了顺式、顺式-环丁烷嘧啶二聚体（CPD）与大肠杆菌 DNA 光解酶结合的情况，考察了温度、酶氧化态、盐和底物构象的影响。尽管结合的总ΔG°相对不受大多数实验条件的影响，但构成结合自由能的焓和熵项对实验条件敏感。底物与 DNA 光解酶的结合通常由焓的负变化驱动。静电相互作用和质子化受所需 FAD 辅因子的氧化态和底物构象的影响。完全还原的酶似乎在结合底物的过程中额外结合了大约两个水分子。更重要的是，实验中热容的变化强烈表明，CPD 损伤必须在与蛋白质结合之前从螺旋内碱基堆积中翻转出来；DNA 修复酶似乎将暴露在溶剂中的 CPD 识别为其损伤识别机制的一部分。
