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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme
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Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

机译：飞秒动力学耦合到Born-Oppenheimer酶中的化学屏障穿越

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摘要

Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nu-cleoside phosphorylase (PNP) forms a ribocation-like transition state in the phosphorolysis of purine nucleosides and fast protein motions have been proposed to participate in barrier crossing. In the present study, ~(13)C-, ~(15)N-, ~2H-labeled human PNP (heavy PNP) was expressed, purified to homogeneity, and shown to exhibit a 9.9% increase in molecular mass relative to its unlabeled counterpart (light PNP). Kinetic isotope effects and steady-state kinetic parameters were indistinguishable for both enzymes, indicating that transition-state structure, equilibrium binding steps, and the rate of product release were not affected by increased protein mass. Single-turnover rate constants were slowed for heavy PNP, demonstrating reduced probability of chemical barrier crossing from enzyme-bound substrates to enzyme-bound products. In a second, independent method to probe barrier crossing, heavy PNP exhibited decreased forward commitment factors, also revealing mass-dependent decreased probability for barrier crossing. Increased atomic mass in human PNP alters bond vibrational modes on the femtosecond time scale and reduces on-enzyme chemical barrier crossing. This study demonstrates coupling of enzymatic bond vibrations on the femtosecond time scale to barrier crossing.
机译：快速（飞秒）动态运动对酶催化位点障碍的贡献是有争议的。人嘌呤核苷磷酸化酶（PNP）在嘌呤核苷的磷酸分解中形成核糖样样过渡状态，并且有人提出快速蛋白质运动参与屏障穿越。在本研究中，〜（13）C-，〜（15）N-，〜2H标记的人PNP（重PNP）被表达，纯化至均质，并且相对于其分子量显示出9.9％的分子量增加未标记的对应物（浅色PNP）。两种酶的动力学同位素效应和稳态动力学参数均无法区分，表明过渡态结构，平衡结合步骤和产物释放速率不受蛋白质质量增加的影响。重PNP的单周转速率常数减慢，表明化学屏障从酶结合的底物穿过酶结合的产物的可能性降低。在第二种独立的探查障碍物穿越方法中，重度PNP表现出降低的前向承诺因子，也揭示了质量依赖性的障碍物穿越概率降低。人类PNP中原子质量的增加会在飞秒时间尺度上改变键的振动模式，并减少酶上化学屏障的穿越。这项研究证明了飞秒时间尺度上的酶键振动与屏障穿越的耦合。

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