Cloning, Overexpression, and Crystallization of Proline Racemase

9407346 Allen这是一项研究计划拨款。计划期间拟进行的研究是脯氨酸消旋酶的克隆、过表达和结晶。计划期之后的研究是用x射线晶体学测定天然状态和底物、底物类似物和过渡状态类似物存在下脯氨酸消旋酶的结构。脯氨酸消旋酶已被证明可以诱导底物的应变/不稳定，这是酶带来速率加速的四种机制之一。诱导菌株在酶-过渡态复合体中被释放。在催化模式中，人们了解最少的是应变/不稳定，因为很难设计实验来评估它。将酶-底物和脯氨酸消旋酶-过渡态类似物的x射线晶体结构进行比较，可以直接观察该酶实现底物应变/不稳定的结构和化学手段。很少有案例证明酶在酶-底物复合体中会发生菌株/不稳定。脯氨酸消旋酶是唯一一个存在过渡态和底物类似物的例子，它适合用x射线晶体学进行研究。这些配合物的结构将为我们理解催化的应变/不稳定模式提供基础。＊＊＊

9407346 Allen This is a Research Planning Grant. The proposed research to be conducted during the planning period is the cloning, overexpression and crystallization of the enzyme proline racemase. The research following the proposed planning period is the determination of the structure of proline racemase by X-ray crystallography in the native state and in the presence of substrates, substrate analogs and transition state analogs. Proline racemase has been shown to induce strain/destabilization of substrate, one of four mechanisms by which enzymes bring about rate accelarations. The induced strain is relieved in the enzyme-transition state complex. Of the modes of catalysis, the least understood is strain/destabilization since it is difficult to devise experiments for its evaluation. The comparison of the X-ray crystal structures of the enzyme-substrate and enzyme- transition state analog complexes of proline racemase will allow direct observation of the structural and chemical means by which this enzyme achieves strain/destabilization of the substrate. %%% There are few cases in which enzymes have been proven to undergo strain/destabilization in the enzyme-substrate complex. Proline racemase is the sole example in which transition state and substrate analogs exist and which is amenable to study by X-ray crystallography. The structure of these complexes will provide the basis by which we can understand the strain/destabilization mode of catalysis. ***