Dynamics in enzymatic catalysis and transition state analogue binding.

我们测量内在动力学同位素效应和解决酶的过渡态（TS）结构的能力 在理解TS的键长、几何形状和静电荷方面取得了重大进展 特定的酶。静电电位图为设计特定的 过渡态类似物，为许多具有fM至pM的Kd值的酶提供TS类似物。TS 分析提供了一个催化，反应物和TS，作为静态对象的两个状态的图片。的应用 punne核苷磷酸化酶（PNP）的计算和实验蛋白质动态测量 乳酸脱氢酶（LDH）揭示了对快速原子运动所需的更深层次的理解 对于化学，TS类似物结合，以及与反应物相关的较慢构象变化 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。 程序项目，并提供一个新的工具，允许前所未有的洞察动态运动， 成本和计算。用具有以下性质的氨基酸取代酶中的天然氨基酸： 增加的质量（2 H，13 C，15 N）改变整个蛋白质（重酶）的原子键频率。 重酶可以通过计算和实验方法来探测，以探索重酶的变化。 键振动频率改变催化性质。人心脏LDH的量子计算预测 在正常酶的过渡态中，氢化物和质子的协同转移， 重酶多动力学同位素效应将解决这些预测实验。重大的人员 PNP显示出比正常酶慢的酶上化学反应，并且计算分析预测了损失 to be related相关to coordinated协调dynamics动态.与施瓦茨，远程突变将被预测，以纠正运动 与TS形成有关。将制作和评价动态工程PNPs。四个循环， PNP催化位点对活性复合物的贡献及其运动将通过 特异性标记、t-跳跃（用Callender）和快速混合（用Dyer）实验。实验和 计算将探索如何优化的TS类似物的人PNP保存蛋白质动力学，而次优 抑制剂冻结某些动态构象。在PNP中使循环变重将探索本地 对非酶化学的贡献。像二氢叶酸还原酶这样的小而重的酶， PNP，表明质量对环路运动的影响。我们将描述三种不同的重酶动力学 通过实验和计算方法的响应。

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.