Tracing enzyme mechanisms across evolution to elucidate the origins of enzymatic catalysis

许多重要的生物学反应自发发生的速度太慢，无法维持生命。酶是自然产生的催化剂，负责将这些反应加速到具有生物学意义的领域。提供的加速通常是深远的，酶催化的反应发生速度比相应的自发过程快1023倍。在一种情况下，一种酶在18毫秒内完成了一个需要8000万年才能完成的反应。虽然这种催化作用的重要性引起了人们的极大关注，但对酶如何如此有效地催化反应的理解仍然难以捉摸。这个项目将通过扩展“酶是如何工作的”这个问题来阐明酶催化的具体决定因素，从而包含一个更大、更有趣的问题：酶是如何进化到如此有效地工作的？通过在进化过程中表征酶的机制，我们的计划将提供与逐渐增加的催化效率相关的具体变化的第一个明确的证明。传统的酶学通过对单一的现代酶的研究来推断酶催化的起源，而我们的项目将引入进化时间的维度来研究酶的机制，以评估对催化很重要的特征。这个程序将提供更多关于酶催化起源的细节，就像电影比静止图像提供更多细节一样。这个提议将使用动力学同位素效应和计算相结合来评估酶是如何在自然和实验室中进化的。自然存在的酶的进化谱系将通过祖先序列重建来确定。这项技术将有效地使已经“死亡”了数十亿年的酶复活，并随后对这些物种的自然谱系进行生化表征。在实验室里，酶可以被迫进化成更有效的催化剂。正如2018年诺贝尔化学奖所显示的那样，这种“定向进化”是一种强大的技术，但它仍然是一个“黑盒子”。我们的项目将展示定向进化导致更高效酶的精确机制，并将改进新型生物催化剂的设计原则。该计划将使用创新的方法来解决酶如何工作的基本问题。我们不能期望有效地设计我们不了解的东西，通过更好地了解酶的工作原理，我们将促进生产具有有用活性的新生物催化剂的努力，这将有利于加拿大的化学工业。这项工作还将加强加拿大作为物理有机化学和酶学领域的领导者的声誉，并将培养出为加拿大科学界学术界和工业界做出贡献的高素质人才。

Many biologically important reactions occur spontaneously at rates that are far too slow to sustain life. Enzymes are the naturally occurring catalysts that are responsible for accelerating these reactions into the realm of biological significance. The accelerations provided are often profound, with enzyme-catalyzed reactions occurring up to 1023-fold faster than the corresponding spontaneous processes. In one case, a reaction that would otherwise require 80 million years is completed by an enzyme in 18 milliseconds. While the magnitude of this catalysis has attracted significant attention, an understanding of how enzymes catalyze reactions so efficiently has remained elusive. This program will elucidate the specific determinants of enzymatic catalysis by extending the question of "how do enzymes work?" to encompass a larger, more interesting one: how do enzymes evolve to work so efficiently? By characterizing enzyme mechanisms over the course of evolution, our program will provide the first unambiguous demonstrations of the specific changes that correlate with gradually increasing catalytic efficiency. Whereas traditional enzymology makes inferences about the origins of enzymatic catalysis from studies of singular, modern enzymes, our program will introduce the dimension of evolutionary time to the study of enzyme mechanisms to evaluate the features that are important to catalysis. This program will provide significantly more detail about the origins of enzymatic catalysis, just as a movie provides more detail than a still image. This proposal will use a combination of kinetic isotope effects and computation to evaluate how enzymes evolve both naturally and in the laboratory. Evolutionary lineages of natural, extant enzymes will be determined using ancestral sequence reconstruction. This technique will effectively permit the resurrection of enzymes that have been "dead" for billions of years and subsequent biochemical characterization of these species along their natural lineages. In the lab, enzymes can be forced to evolve into more efficient catalysts. This "directed evolution" is a powerful technique, as shown by the 2018 Nobel Prize in Chemistry, but it remains a "black box". Our program will demonstrate the precise mechanisms by which directed evolution leads to more efficient enzymes and will lead to improved design principles for novel biocatalysts. This program will use an innovative approach to address the fundamental question of how enzymes work. We cannot expect to efficiently engineer what we do not understand, and by better understanding how enzymes work, we will facilitate efforts to produce de novo biocatalysts with useful activities that will benefit the Canadian chemical industry. This work will also strengthen Canada's reputation as a leader in the fields of physical organic chemistry and enzymology and will produce HQP who will be well prepared to contribute to the Canadian scientific community in academia and industry.