基于脂肪酶A突变体探索无固定结构蛋白催化机制

In Traditional opinion, the stability of the advanced structure of protein is necessary for its function in biosystem. Recently, it's found that there were a large number of proteins with disordered state in the organism, and their special "folding on binding" mechanism attracted the interests from the scientific communities. And also, it's found that the catalytic functions of enzyme could be altered from specifity to versatility along with the change of the condition. and thus, it's deduced that the enzyme could possess various catalytic structures toward the different substrates. From this, we proposed a new mechanism, "catalysis with folding on binding", which described the catalytic process of enzyme. In this respect, we plan to deploy a series of further studies, that is, (ⅰ) construct a model enzyme by the molecular biology technique. (ⅱ) identify the reaction pathway and the geometries and energy of the stationary points (intermediates, transition states) via the theoretical simulation, and clarify the influence of the disordered state towards the catalytic activity and the properties of disordered protein.(ⅲ)the site-directed mutation was employed to verify and optimize the theoretical results.

传统认为，稳定的高级结构是蛋白质要发挥功能的必要条件。近年来，人们发现生物中存在大量的无稳定结构的蛋白质,且它们独特的"折叠偶合结合"作用机理引起了科学共同体的关注。同时，申请人经过长期研究发现，一些酶随着条件的变化，其催化功能专一性变成的多功能性。由此推测，随着条件变化，酶分子的结构相对于不同的底物分子呈现出不同的催化结构，从而我们提出了一种新的酶催化机理"折叠-结合偶合催化"，我们预开展如下研究工作，1通过分子生物学技术构建一个模型酶；2借助理论模拟等手段确定反应路径和反应中各驻点（中间体与过渡态）构型与能量，最终得到整个反应的势能变化剖面，详细阐述无固定结构肽段对酶催化反应性质和活性的影响。3运用定点突变技术验证和优化理论研究结果。

本研究首先提出了一个新的酶催化机制-“折叠-结合偶合催化”新的催化机制。为此，为了验证这个机制，我们在影响酶的各种因素方面展开了一系列的研究。首先我们通过底物小分子库对酶与底物识别进行实验研究，然后通过QSAR技术进行统计分析总结得出规律。其次，通过分子动力学及QM/MM方法研究酶中各种非键作用的功能及对酶催化活性的影响和通过对酶活性中心残基能量贡献评估确定重要残基。再者，分别用实验和理论考察无规序列的在酶与底物分子识别、结合及催化过程中的结构变化。我们发现，首先，酶能识别一系列化学结构相近的小分子底物，从而说明酶的活性中心残基所构成的高级结构对不同的小分子具有一定的适应性。其次，由残基能量贡献评估可知，构成酶催化中心的高级结构残基具有相当高可替代性。再者，酶催化中，无规片段的在不同底物诱导下，能产生不同类型的结构，但此结构稳定性较差。