Thermodynamic analysis of small ligand binding to the Escherichia coli repressor of biotin biosynthesis.
Thermodynamic analysis of small ligand binding to the Escherichia coli repressor of biotin biosynthesis.
复制标题
与生物素生物合成的大肠杆菌阻遏物结合的小配体的热力学分析。
DOI：
10.1021/bi9600658
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Beckett,D
中科院分区：
文献类型：
--
作者：
Xu,Y;Johnson,CR;Beckett,D
BIRA是生物素生物合成的转录抑制因子，也是生物素全酶合成酶。它催化以生物素和三磷酸腺苷为底物合成生物素-5‘-AMP。腺苷是生物素转移反应中的活性中间体，也是位点特异性DNA结合的正变构效应。BIRA对腺苷的亲和力比它对生物素的亲和力大得多，这两种结合反应都与蛋白质构象的变化有关。用动力学方法测定了这两个结合作用的温度依赖性，由动力学数据得到的平衡离解常数的Van‘t Hoff分析表明，虽然这两个结合过程的特征是大的负焓，但对两个过程的熵贡献都很小。用等温滴定量热法测定了结合热。与van‘t Hoff分析的结果一致，量热焓较大且为负值。量热测量的更高精度允许更准确地估计对结合过程的熵贡献，这对两个配体具有相反的符号。此外，与这两个结合反应相关的热容变化很小。利用测得的生物素和BIO-5‘-AMP与BIRA结合的热力学参数，分析了它们对结合能的结构贡献。这些计算的结果表明，极地和非极区表面积的埋藏对这两个结合过程的贡献相当。然而，对于生物素结合，溶剂可及表面积的总损失更大。分析进一步表明，虽然这两个结合反应都与构象熵的损失有关，但生物素结合的构象变化幅度要大得多。
BirA is the transcriptional repressor of biotin biosynthesis and a biotin holoenzyme synthetase. It catalyzes synthesis of biotinyl-5‘-AMP from the substrates biotin and ATP. The adenylate is the activated intermediate in the biotin transfer reaction as well as the positive allosteric effector for site-specific DNA binding. The affinity of BirA for the adenylate is considerably greater than its affinity for biotin, and both binding reactions are coupled to changes in the conformation of the protein. The temperature dependencies of the two binding interactions have been determined using kinetic techniques.Van't Hoff analyses of the equilibrium dissociation constants derived from the kinetic data indicate that while the two binding processes are characterized by large negative enthalpies, the entropic contributions are small for both. Binding enthalpies have also been determined by isothermal titration calorimetry. Consistent with the results of the van't Hoff analyses, the calorimetric enthalpies are large and negative. The greater precision of the calorimetric measurements allowed more accurate estimation of the entropic contributions to the binding processes, which are of opposite sign for the two ligands. In addition, the heat capacity changes associated with the two binding reactions are small. The measured thermodynamic parameters for binding of biotin and bio-5‘-AMP to BirA have been utilized to dissect out structural contributions to the binding energetics. Results of these calculations indicate equivalent contributions of burial of polar and apolar surface area to both binding processes. The total loss of solvent accessible surface area is, however, greater for biotin binding. The analysis indicates furthermore that although both binding reactions are coupled to losses in configurational entropy, the magnitude of the conformational change is significantly larger for biotin binding.