Dynamics in enzymatic catalysis and transition state analogue binding.

我们测量本征动力学同位素效应和解决酶过渡态(TS)结构的能力 在理解TSS的键长、几何形状和静电电荷方面取得了重大进展 一种特定的酶。TSS的静电势图为具体的设计提供了蓝图 过渡态类似物，为许多酶提供具有Fm到Pm的Kd值的TS类似物TS 分析提供了催化剂、反应物和TSS作为静态对象的两种状态图。技术的应用 双核核苷磷酸化酶(PNP)的计算和扩展蛋白质动力学测量 乳酸脱氢酶(LDH)揭示了对所需的快速原子运动的更深层次的理解 对于化学，TS模拟结合，以及与反应物相关的较慢的构象变化 结合、催化位点重组和产物释放。重酶是最近在这方面的先驱 计划项目并提供一种新工具，允许对动态运动进行前所未有的深入了解 在费用上和计算上。用天然氨基酸取代酶中的天然氨基酸 质量的增加(2H、13C、15N)改变了整个蛋白质(重酶)的原子键频率。 可以通过计算和实验方法来探索重酶是如何变化的 键的振动频率会改变催化性能。量子计算与人体心脏乳酸脱氢酶预测 在正常酶中氢化物和质子转移处于过渡态，而在正常酶中顺序转移 重酵素。多重动力学同位素效应将在实验上解决这些预测。笨重的人类 PNP显示出比正常酶更慢的酶上化学，计算分析预测了这种损失 与协调动力有关。有了施瓦茨，远程突变将被预测为纠正运动 与TS地层有关。将生产和评估动态设计的PNPs。四个环路在 PNP催化中心与活性络合物的含量和它们的运动将分别通过 具体的标签，t跳跃(使用Callender)和快速混合(使用Dyer)实验。实验和 计算将探索优化的人类PNP TS类似物如何在次优的情况下保持蛋白质动力学 抑制剂冻结了某些动态构象。使循环在PnP中变得繁重将探索局部 对酶化学的贡献。小而重的酶，如二氢叶酸还原酶，其作用与 PnP，暗示了环路运动的质量效应。我们将描述三种不同的重酶动力学 通过实验和计算方法作出反应。

Our ability to measure intrinsic kinetic isotope effects and solve enzymatic transition state (TS) structures provided a major advance in understanding the bond lengths, geometry and electrostatic charges of the TSs of specific enzymes. Electrostatic potential maps of TSs provided blueprints for the design of specific transition state analogues, providing TS analogues for many enzymes with Kd values of fM to pM. TS analysis provides a two-state picture of catalysis, reactants and TSs, as static objects. The application of computational and expenmental protein dynamic measurements to punne nucleoside phosphorylase (PNP) and lactate dehydrogenase (LDH) is revealing a deeper understanding of the fast atomic motions required for chemistry, TS analogue binding, and the slower conformational changes associated with reactant binding, catalytic site reorganization and product release. Heavy enzymes were recently pioneered in this program project and provide a new tool permitting unprecedented insight into dynamic motion both expenmentally and computationally. Replacing natural amino acids in enzymes with those having increased mass (2H, 13C, 15N) changes atomic bond frequencies throughout the protein (heavy enzyme). Heavy enzymes can be probed by computational and experimental approaches to explore how changes in bond vibrational frequency alter catalytic properties. Quantum calculations with human heart LDH predict concerted hydride and proton transfer in the transition state in normal enzyme but sequential transfer in the heavy enzyme. Multiple kinetic isotope effects will resolve these predictions experimentally. Heavy human PNP shows slower on-enzyme chemistry than normal enzyme and computational analysis predicts the loss to be related to coordinated dynamics. With Schwartz, remote mutations will be predicted to correct motions associated with TS formation. Dynamically engineered PNPs will be produced and evaluated. Four loops at the PNP catalytic site contnbute to the active complex and their motions will be individually monitored by specific labels, t-jump (with Callender) and rapid mixing (with Dyer) experiments. Experiments and computation will explore how optimized TS analogues of human PNP conserve protein dynamics while sub-optimal inhibitors freeze certain dynamic conformations. Making loops heavy in PNP will explore local contributions to on-enzyme chemistry. Small, heavy enzymes like dihydrofolate reductase act differently from PNP, suggesting mass effects on loop motion. We will characterize three distinct heavy enzyme dynamic responses by experimental and computational approaches.