Dynamics, Function, and Stability in Large Enzymes

描述（由申请人提供）：在酶促反应过程中发生许多构象转变。表征这些动力学过程的物理细节对于理解酶催化、蛋白质/配体相互作用和蛋白质能量景观中的波动是必不可少的。迄今为止，进行的有限数量的研究已经解决了低分子量的酶或蛋白质中的这些问题。大多数酶比非催化蛋白质大，有证据表明大蛋白质的动力学和能量特性与小蛋白质不同，因此目前的理解可能不适用于其他系统。此外，正确评估功能和动力学之间的相互作用需要脱离单一实验技术的限制。因此，动力学和功能的作用将解决使用溶液NMR光谱，配体合成，生化表征和计算。本提案中描述的研究集中在大酶，VanX（46 kDa），一种细菌对抗生素万古霉素和磷酸丙糖异构酶（TIM，54 kDa）的耐药性所必需的酶，一种功能障碍与非球形细胞溶血性贫血和神经系统疾病相关的模型酶。催化和稳定性的结构，动态和充满活力的贡献将通过研究VanX和TIM的配合物，模仿反应途径中的离散步骤。本申请的具体长期目标是确定VanX的结构和构象变化，这些变化导致其催化活性的抑制以及其对酰胺水解的底物偏好超过酯。对于TIM，将通过NMR自旋弛豫测量、定点突变和计算研究来研究最佳催化的运动事件的适当时机。实验的这种组合将允许表征催化重要残基和活性位点环的动力学。

DESCRIPTION (provided by applicant): Many conformational transitions transpire during the course of an enzymatic reaction. Characterization of the physical details of these dynamical processes is essential for understanding enzyme catalysis, protein/ligand interactions, and fluctuations in the protein energy landscape. To date, the limited number of studies performed has addressed these issues in enzymes or proteins of low molecular weight. The majority of enzymes are larger than non-catalytic proteins and there is evidence that the dynamic and energetic properties of large proteins are distinct from smaller ones, therefore current understanding may not apply to other systems. In addition, the proper assessment of the interplay between function and dynamics requires a departure from the restraints of a single experimental technique. Accordingly, the role of dynamics and function will be addressed using solution NMR spectroscopy, ligand synthesis, biochemical characterization, and computation. The studies described in this proposal focus on large enzymes, VanX (46 kDa) an enzyme essential for bacterial resistance to the antibiotic vancomycin and triosephosphate isomerase (TIM, 54 kDa), a model enzyme whose dysfunction is associated with nonspherocytic hemolytic anemia and neurological disorders. The structure, dynamic, and energetic contributions to catalysis and stability will be addressed through studies of VanX and TIM in complexes that mimic discreet steps in the reaction pathway. The specific long-term goals of this application are to determine the structure and conformational changes in VanX that lead to inhibition of its catalytic activity and to its substrate preference for hydrolysis of amides over esters. For TIM, the proper timing of motional events for optimal catalysis will be investigated by NMR spin-relaxation measurements, site-directed mutation, and computational studies. This combination of experiments will allow characterization of the dynamics of catalytically important residues and the active site loop.