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。世纪中期的许多生物化学研究都集中在识别生物学中发现的个体反应以及这些反应如何共同形成代谢途径。随着这些反应和途径的发现，焦点转移到负责每个反应的特定酶。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描述酶催化反应的化学转化，涉及酶结合的辅因子（通常是维生素或衍生自维生素）和特定的氨基酸侧链，以促进某些类型的化学转化。世纪后半叶，酶学的大部分工作集中在确定酶催化的各种转化的化学途径上。这一阶段的生物化学和酶学研究非常成功。第二个基本问题是：酶使用什么机制来实现其巨大的速率增强和精致的特异性？关于酶的催化能力起源的推测和模型伴随着上述研究，但没有进展到接近化学机制的程度。重要的概括已经作出，如使用结合能从酶活性位点内的相互作用，以促进催化，从而连接特异性和催化。然而，有多种酶催化的竞争描述。衡量一个学术领域封闭程度的一个标准是教科书中对该领域的描述。对于酶特异性和速率增强的机制，不同文献的处理方法差异很大，而且往往提供关于特定酶的轶事信息或模型名称，而没有明确的解释。由于过去几十年来已经开发出了强大的工具，这一领域已经成熟，可以取得进展。该项目将利用传统的方法，但也联合收割机与其他化学，物理和计算工具相结合。这样一个基础广泛的方法将需要获得酶的能量学和物理特性的综合理解，这反过来又需要集中精力在一个系统，是经得起这样的攻击。先前的研究已经表明，酶酮甾体异构酶（KSI）是理想的;它是非常容易处理的，适合于过多的方法。KSI使用的两种催化模式和许多酶的共同点将被探索：一般酸/碱催化（GABC）和过渡态互补性（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，以增加代表性不足的群体的参与。将为高中生举办一个讲习班，其中包括与研究生的互动和讨论。这些讲习班旨在告知对科学有明确兴趣的学生如何进行科学职业培训。学生将通过该计划获得建议和指导，以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编制促进对各级学生的培训。

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.