Cloning, Overexpression, and Crystallization of Proline Racemase

小艾伦9407346 这是一个研究规划补助金。 拟议在规划期间进行的研究是脯氨酸消旋酶的克隆、过度表达和结晶。 拟议规划期之后的研究是通过X射线晶体学测定脯氨酸消旋酶在天然状态下以及在底物、底物类似物和过渡态类似物存在下的结构。 脯氨酸消旋酶已显示出诱导底物的应变/去稳定化，这是酶引起速率加速的四种机制之一。诱导应变在酶-过渡态复合物中被缓解。 在催化的模式中，最不了解的是应变/去稳定化，因为很难设计实验对其进行评估。 脯氨酸消旋酶的酶-底物和酶-过渡态类似物复合物的X射线晶体结构的比较将允许直接观察该酶实现底物的应变/去稳定化的结构和化学手段。 在少数情况下，酶已被证明在酶-底物复合物中经历应变/去稳定化。 脯氨酸消旋酶是存在过渡态和底物类似物的唯一例子，并且可以通过X射线晶体学进行研究。 这些配合物的结构将提供基础，通过它我们可以理解的应变/不稳定模式的催化。 ***

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. ***