Xu Y, Johnson C R, Beckett D
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore 21228, USA.
Biochemistry. 1996 Apr 30;35(17):5509-17. doi: 10.1021/bi9600658.
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 analysis 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.
BirA是生物素生物合成的转录阻遏物和生物素全酶合成酶。它催化从底物生物素和ATP合成生物素基-5'-AMP。腺苷酸是生物素转移反应中的活化中间体，也是位点特异性DNA结合的正别构效应物。BirA对腺苷酸的亲和力远大于其对生物素的亲和力，并且两种结合反应都与蛋白质构象的变化相关联。已使用动力学技术确定了两种结合相互作用的温度依赖性。对由动力学数据得出的平衡解离常数进行范特霍夫分析表明，虽然两种结合过程的特征是具有很大的负焓，但两者的熵贡献都很小。结合焓也已通过等温滴定量热法测定。与范特霍夫分析的结果一致，量热焓很大且为负。量热测量的更高精度使得能够更准确地估计结合过程中的熵贡献，这两种配体的熵贡献符号相反。此外，与两种结合反应相关的热容变化很小。用于分析生物素和生物-5'-AMP与BirA结合的测量热力学参数已被用于剖析结合能的结构贡献。这些计算结果表明，极性和非极性表面积的埋藏对两种结合过程的贡献相当。然而，生物素结合时溶剂可及表面积的总损失更大。分析还表明，虽然两种结合反应都与构象熵的损失相关，但生物素结合时构象变化的幅度明显更大。
