Kladova O A, Kuznetsov N A, Fedorova O S
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Akad. Lavrentiev Ave. 8, 630090, Novosibirsk, Russia.
Department of Natural Sciences, Novosibirsk State University, Pirogova Str. 2, 630090, Novosibirsk Russia.
Acta Naturae. 2019 Jan-Mar;11(1):29-37.
In the present work, a thermodynamic analysis of the interaction between endonuclease VIII (Endo VIII) and model DNA substrates containing damaged nucleotides, such as 5,6-dihydrouridine and 2-hydroxymethyl-3-hydroxytetrahydrofuran (F-site), was performed. The changes in the fluorescence intensity of the 1,3-diaza-2-oxophenoxazine (tC°) residue located in the complementary chain opposite to the specific site were recorded in the course of the enzyme-substrate interaction. The kinetics was analyzed by the stopped-flow method at different temperatures. The changes of standard Gibbs free energy, enthalpy, and entropy of sequential steps of DNA substrate binding, as well as activation enthalpy and entropy for the transition complex formation of the catalytic stage, were calculated. The comparison of the kinetic and thermodynamic data characterizing the conformational transitions of enzyme and DNA in the course of their interaction made it possible to specify the nature of the molecular processes occurring at the stages of substrate binding, recognition of the damaged base, and its removal from DNA.
在本研究中，对核酸内切酶VIII（Endo VIII）与含有受损核苷酸的模型DNA底物（如5,6 - 二氢尿苷和2 - 羟甲基 - 3 - 羟基四氢呋喃（F位点））之间的相互作用进行了热力学分析。在酶 - 底物相互作用过程中，记录了位于与特定位点互补链上的1,3 - 二氮杂 - 2 - 氧代吩恶嗪（tC°）残基荧光强度的变化。通过停流法在不同温度下分析动力学。计算了DNA底物结合连续步骤的标准吉布斯自由能、焓和熵的变化，以及催化阶段过渡复合物形成的活化焓和熵。对表征酶和DNA在相互作用过程中构象转变的动力学和热力学数据进行比较，使得确定在底物结合、受损碱基识别及其从DNA中去除阶段发生的分子过程的性质成为可能。
