Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18661-5. doi: 10.1073/pnas.1114900108. Epub 2011 Nov 7.
Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nucleoside 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-, (2)H-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.
快速（飞秒）动态运动对酶催化位点的跨越障碍的贡献存在争议。人类嘌呤核苷磷酸化酶（PNP）在嘌呤核苷的磷酸解中形成类似核糖离子的过渡态，并且已经提出快速蛋白质运动参与了障碍跨越。在本研究中，表达、纯化为均相的（13）C、（15）N、（2）H 标记的人 PNP（重 PNP），与未标记的 PNP（轻 PNP）相比，其分子量增加了 9.9%。两种酶的动力学同位素效应和稳态动力学参数均无明显差异，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 的单轮反应速率常数减慢，表明从酶结合底物到酶结合产物的化学障碍跨越的可能性降低。在第二种独立的探测障碍跨越的方法中，重 PNP 表现出降低的前向承诺因子，也揭示了障碍跨越的概率因质量依赖性而降低。人 PNP 的原子质量增加会在飞秒时间尺度上改变键振动模式，并降低酶上的化学障碍跨越。这项研究表明，酶键振动的耦合在飞秒时间尺度上与障碍跨越有关。
