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

追踪进化过程中的酶机制以阐明酶催化的起源

• 批准号: RGPIN-2020-04455
• 负责人: Howe, Graeme
• 金额: $ 2.11万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=747912
• 关键词: Tracing; enzyme; mechanisms; across; evolution

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.

许多生物学上重要的反应自发发生的速度太慢，无法维持生命。酶是天然存在的催化剂，负责加速这些反应进入生物学意义的领域。所提供的加速通常是深刻的，酶催化反应的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8000万年的反应。虽然这种催化作用的大小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。这个程序将阐明酶催化的具体决定因素，通过扩展的问题“酶如何工作？“来涵盖一个更大、更有趣的问题：酶是如何进化到如此有效地工作的？通过表征进化过程中的酶机制，我们的计划将提供与逐渐增加的催化效率相关的特定变化的第一个明确的证明。传统的酶学从对单一的现代酶的研究中推断酶催化的起源，而我们的计划将引入进化时间的维度来研究酶机制，以评估对催化重要的特征。这个程序将提供关于酶催化起源的更多细节，就像电影比静止图像提供更多细节一样。 该提案将使用动力学同位素效应和计算的组合来评估酶如何在自然和实验室中进化。天然的、现存的酶的进化谱系将使用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，并使这些物种沿着它们的自然谱系进行后续的生物化学表征。在实验室中，酶可以被迫进化成更有效的催化剂。正如2018年诺贝尔化学奖所展示的那样，这种“定向进化”是一种强大的技术，但它仍然是一个“黑匣子”。我们的计划将展示定向进化导致更有效的酶的精确机制，并将导致新型生物催化剂的改进设计原则。 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从头生物催化剂的努力，这将有利于加拿大化学工业。这项工作还将加强加拿大作为物理有机化学和酶学领域领导者的声誉，并将产生HQP，他们将为加拿大学术界和工业界的科学界做出贡献做好充分准备。