Heavy enzymes: Probing fast dynamics in enzyme catalysis by mass modulation

生物化学的一个中心范式是蛋白质的功能是由结构决定的。然而，在溶液中，蛋白质本质上是动态分子，在时间尺度上表现出从键拉伸到慢域运动和正常模式振动的运动。酶学中一个重要的开放性问题仍然是这种动力学的作用，以及在时间尺度上比周转率更快的运动/振动是否可以在催化过程中耦合到化学步骤。在利用核磁共振方法确定较慢动力学（如酶转换过程中的环开/关）的作用方面已经取得了很好的进展，但是更快（亚纳秒）动力学与化学耦合的直接证据仍然是模糊的和有争议的，并且主要基于动力学同位素效应（KIE，例如速率常数之比：kH/kD）的异常温度依赖性。这种快速动力学的作用仍然是一个重要的问题，因为在与化学相似的时间尺度上的运动有可能深刻地影响反应结果，从而提供一种控制（酶）反应性的方法。稳定的同位素标记蛋白质（通常含有2H、13C和/或15N）作为一种实验工具已经被开发了很多年，特别是核磁共振和振动光谱社区。通常隐含的假设是，同位素标记不会显著干扰蛋白质的功能。然而，研究表明，一些同位素标记的“重”酶具有可测量的较慢的反应动力学。这些数据是根据“Born-Oppenheimer近似”来解释的，其中增加的蛋白质质量（由于标记）改变了键振动频率，而不影响酶的静电特性。在这种情况下，这些结果表明，“重酶”中较低频率的（快速）键振动可能导致构象采样减少，从而导致化学屏障穿越；反应的速率与越过势垒的速率成正比。我们最近扩展了“重酶”方法来研究老黄酶（OYE）季戊四醇四硝酸盐还原酶（PETNR）的振动耦合。我们发现，KIE的温度依赖性在“重”PETNR中显著增加，KIE通常被用作蛋白质环境偶联的决定性证据。这强烈表明，振动耦合可以通过对蛋白质进行同位素标记来增强。显然，“重酶”方法可以作为研究酶偶联和动力学的有力工具，但重要的问题仍然存在。质量扰动将影响蛋白质内部的所有振动，因此，为了牢固地建立“重酶”效应的理论起源，对蛋白质和化学坐标之间任何相关振动耦合的时间尺度的实验观察是非常可取的。此外，对“重”DHFR酶的计算研究表明，与化学配位的动态耦合增加对DHFR催化是有害的。现在是时候考虑酶运动与化学坐标的动态耦合是否通常被优化（例如通过进化），从而是否可以利用它来增强反应性或“药物”酶靶标。通过将我们独特的变温变压KIE测量方法与“重”酶方法相结合，我们将研究两种同源OYEs， PETNR和吗啡酮还原酶（MR）在催化循环的两半中的H转移反应。选择性辅因子和氨基酸标记我们将识别（如果存在的话）振动耦合残基网络，而分子动力学模拟和超快光谱将用于建立这种耦合振动的时间尺度。

A central paradigm in biochemistry is that protein function is defined by structure. However, in solution proteins are inherently dynamic molecules, exhibiting motions on timescales ranging from bond stretches through to slow domain motions and normal mode vibrations. An important open question in enzymology remains the role of such dynamics, and whether motions/vibrations on timescales faster than turnover can couple to chemical steps during catalysis. There has been good progress in establishing the role of slower dynamics such as loop opening/closing during enzyme turnover using NMR approaches, but direct evidence for the coupling of faster (sub-nanosecond) dynamics to chemistry remains illusive and controversial and is based largely on the anomalous temperature dependencies of kinetic isotope effects (KIE; e.g. the ratio of rate constants: kH/kD). The role of such fast dynamics remains an important question, as motions on similar timescales to chemistry have the potential to profoundly affect the reaction outcome, and thus offer a means to control (enzyme) reactivity. Stable isotopically-labelled proteins (typically with 2H, 13C and/or 15N) have been exploited as an experimental tool for many years, particularly by the NMR and vibrational spectroscopy communities. The implicit assumption has generally been that isotopic labelling does not significantly perturb protein function. However, it was demonstrated that several isotopically labelled 'heavy' enzymes have measurably slower reaction kinetics. These data were interpreted in terms of the 'Born-Oppenheimer approximation', where increased protein mass (due to labelling) alters bond vibrational frequencies without affecting electrostatic properties of the enzyme. In this case, these results suggest that the lower frequency of (fast) bond vibrations in the 'heavy enzymes' may lead to a reduction in conformational sampling and thus chemical barrier crossing; the rate of reaction is proportional to the rate of barrier crossing. We recently extended the 'heavy enzyme' approach to study vibrational coupling in the Old Yellow enzyme (OYE) pentaerythritol tetranitrate reductase (PETNR). We showed that the temperature dependence of the KIE, which is often used as definitive evidence of protein environmental coupling, is significantly increased in 'heavy' PETNR. This strongly suggests that vibrational coupling can be enhanced by isotopic labelling of proteins. Clearly, the 'heavy enzyme' methodology can be used as a powerful tool to study enzyme coupling and dynamics, but important questions remain. Mass perturbation will affect all vibrations within the protein, so experimental observation of the timescale(s) of any relevant vibrational coupling between protein and chemical coordinate is highly desirable in order to firmly establish the theoretical origin of the 'heavy enzyme' effect. Further, a computational study of a 'heavy' DHFR enzymes suggests that an increased dynamic coupling to the chemical coordinate is detrimental to DHFR catalysis. It is now timely to also consider whether the dynamic coupling of enzyme motions to the chemical coordinate is generally optimised (e.g. by evolution) and thus whether this could be exploited to enhance reactivity or 'drug' enzyme targets.By combining our unique variable temperature and pressure KIE measurements with the 'heavy' enzyme method, we will study the H transfer reactions during both halves of the catalytic cycles of two homologous OYEs, PETNR and morphinone reductase (MR). Selective cofactor and amino acid labelling we will identify (if present) networks of vibrationally-coupled residues, while molecular dynamics simulations and ultrafast spectroscopies will be used to establish the timescales of such coupled vibrations.