Dynamics, Function, and Stability in Large Enzymes

描述(由申请人提供)：在酶反应过程中会发生许多构象转变。这些动态过程的物理细节的表征对于理解酶催化、蛋白质/配体相互作用和蛋白质能量格局中的波动是至关重要的。到目前为止，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。大多数酶都比非催化蛋白质大，有证据表明大蛋白质的动态和能量性质与小蛋白质不同，因此目前的理解可能不适用于其他系统。此外，对功能和动力学之间相互作用的适当评估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通过溶液核磁共振光谱学、配体合成、生化表征和计算来解决。本提案中描述的研究重点是大型酶VanX(46 KDa)，这是细菌对抗生素万古霉素耐药所必需的酶和磷酸丙糖异构酶(TIM，54 kDa)，这是一种模型酶，其功能障碍与非球形细胞溶血性贫血和神经疾病有关。结构、动力学和能量对催化和稳定性的贡献将通过对VANX和TIM在模拟反应路径中的谨慎步骤的络合物中的研究来解决。这项应用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被抑制，并导致其对酰胺类水解的底物优先于酯。对于TIM来说，最佳催化的运动事件的适当时机将通过核磁共振自旋弛豫测量、定点突变和计算研究来研究。这一组合的实验将允许表征具有催化重要性的残基和活性部位环的动力学。

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.