Mechanistic Investigations of Ketosteroid Isomerase

酶是细胞内的大分子，可以加速生命所需的特定化学转化。20世纪中叶的大部分生物化学研究集中于确定生物学中发现的个别反应，以及这些反应是如何结合在一起形成代谢途径的。随着这些反应和途径的发现，焦点转移到了负责每个反应的特定酶上。这项工作的大部分涉及必要的基本表征，许多研究人员的研究能够从化学转化的角度描述酶催化的反应，这些化学转化涉及酶结合的辅因子(通常是维生素或从维生素衍生的)和特定的氨基酸侧链，以促进某些类型的化学转化。20世纪下半叶，酶学的大部分工作集中在确定这些由酶催化的各种转化的化学途径。生物化学和酶学的这一阶段的研究已经非常成功。第二个基本问题是：酶用什么机制来实现其巨大的速率增强和精致的特异性？关于酶催化能力起源的推测和模型伴随着上述研究，但没有取得化学机制所具有的近乎封闭的结果。已经做出了重要的概括，例如利用酶活性部位之间相互作用的结合能来促进催化，从而将特异性和催化联系起来。然而，关于酶催化的描述是多方面的、相互竞争的。衡量学术领域封闭程度的一个指标是教科书中对该领域的描述。对于酶的专一性和速率增强的机制，不同文本的处理方法差别很大，通常提供关于特定酶的轶事信息或模型的名称，而没有明确的描述。这个项目的一个目标是提供对酶如何实现其速率增强的深入和基本的理解。由于过去几十年来开发的强大工具，这一领域取得进展的时机已经成熟。该项目将利用传统方法，但也将它们与其他化学、物理和计算工具相结合。需要这样一种基础广泛的方法来实现对酶、能量学和物理性质的综合理解，而这反过来又需要将重点放在一个能够承受这种攻击的系统上。先前的研究表明，酮类类固醇异构酶(KSI)是一种理想的酶，它非常容易处理，并适用于太多的方法。将探索KSI使用的和许多酶共同使用的两种催化模式：一般酸/碱催化(GABC)和过渡态络合(通过所谓的氧阴离子空穴)。该项目的一个重要组成部分是向高中生介绍科学职业，以增加未被充分代表的群体的广泛参与。将为高中生发起一个研讨会，其中将包括与研究生的互动和讨论。这些工作坊的目的是让对科学有明显兴趣的学生了解如何为科学生涯进行培训。通过这个项目，学生将得到建议和指导，培养学生对科学的兴趣。该项目将继续通过实验研究和课程开发促进各级学生的培训。

Enzymes are the macromolecules within cells that accelerate the specific chemical transformations needed for life. Much of biochemistry research in the mid-20th century focused on identifying the individual reactions that are found in biology and how these reactions come together to make metabolic pathways. As these reactions and pathways were discovered, focus shifted to the specific enzymes responsible for each reaction. Much of this work involved necessary basic characterization, and studies by many investigators were able to describe enzyme-catalyzed reactions in terms of chemical transformations that involve enzyme-bound cofactors (which are often vitamins or derived from vitamins) and specific amino acid side chains to facilitate certain types of chemical transformations. The majority of work in enzymology in the second half of the 20th century focused on determining these chemical pathways for the various transformations catalyzed by enzymes. This phase of research in biochemistry and enzymology has been highly successful.A second fundamental question is: What are the mechanisms that enzymes use to achieve their enormous rate enhancements and exquisite specificities? Speculations and models about the origin of the catalytic power of enzymes accompanied the above studies, but did not advance to attain the near-closure that the chemical mechanisms have. Important generalizations have been made, such as the use of binding energy from interactions within enzyme active sites to facilitate catalysis, thereby linking specificity and catalysis. Nevertheless, there are multiple, competing descriptions of enzymatic catalysis. One measure of the degree of closure in an academic area is the descriptions of that area in textbooks. For the mechanisms of enzyme specificity and rate enhancements, the treatments vary greatly from text to text and often provide present anecdotal information about particular enzymes or the names of models without clear description.A goal of this project is to provide in-depth and fundamental understanding of how enzymes achieve their rate enhancements. This field is ripe for advances because of the powerful tools that have been developed over the past decades. The project will utilize traditional approaches but also combine them with additional chemical, physical, and computational tools. Such a broad-based approach will be required to attain an integrated understanding of enzyme energetics and physical properties and this in turn will require intense focus on a system that is amenable to such an assault. Prior research has shown that the enzyme ketosteroid isomerase (KSI) is ideal; it is highly tractable and amenable to plethora of approaches. Two modes of catalysis that are used by KSI and common to many enzymes will be explored: general acid/base catalysis (GABC) and transition state complemetarity (via a so-called oxyanion hole).An important component of this project is introducing high school students to careers in science to increase the broadening participation of underrepresented groups. A workshop will be initiated for high school students that will include interactions and discussions with graduate students. These workshops are intended to inform students with a clear interest in science how to train for a scientific career. The students will receive advice and mentorship through this program to cultivate the students interest in science. The project will continue to promote the training of students at all levels through experimental research and curriculum development.