Collaborative Research of Proton Transfers in Enzymes: A Synergetic Theory-Experiment Approach

酶中质子转移的合作研究：理论-实验协同方法

• 批准号: 0313743
• 负责人: Hua Guo
• 金额: $ 17.16万
• 依托单位: University of New Mexico
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0313743&HistoricalAwards=false
• 关键词: Collaborative; Research; Proton; Transfers; Enzymes

This collaborative project involves a combined theoretical-experimental approach that aims at a better understanding of proton transfer processes in enzymes. A specific focus will be placed on the carbonic anhydrase family, in which intramolecular proton transfer is the rate-limiting step. To guide the experimental design of new and more efficient proton transfer pathways in carbonic anhydrase, a series of calculations will be carried out using QM/MM methods based on a recently proposed approximate density functional theory (SCC-DFTB) and the CHARMM force field. These calculations will provide microscopic information about the free energy barrier, rate constant, solvent effect, kinetic isotope effect, and more importantly the contribution of individual residues to the proton transfer process. On the experimental side, site-directed mutagenesis of selected active site residues of the enzyme will be carried out to introduce chemically modified amino acids containing artificial proton shuttles; the location and nature of these groups will be guided by computational studies. Structural measurements of modified enzymes using x-ray crystallography will yield valuable information about the spatial organizations of the active site, such as the orientation of the new shuttle group and its distance to the Zn ion as well as the configuration of water bridges between the proton donor and acceptor groups. Kinetic studies, utilizing isotopic substitutions and pH changes, will reveal the catalytic effectiveness of the mutation(s). The strength of the present project lies in a coherent interplay between state-of-the-art experimental and theoretical investigations. Experimental results help to define the key questions for theoretical simulations, while in turn theoretical studies provide both interpretation of experimental findings and guidance to further experimental exploration.Broader Impact: In addition to providing mechanistic insights into the function of carbonic anhydrase, the present project has broader impacts on the understanding of proton transfers in other enzyme systems and on uncovering general principles for de novo protein engineering. The research will also provide ample opportunities for participating students to acquire important skills in computational chemistry, organic and biological chemistry, molecular biology, and structural biology. Finally, the integrated theoretical-experimental research will be invaluable for motivating undergraduate students and attracting them to scientific research and discovery.

该合作项目涉及理论与实验相结合的方法，旨在更好地了解酶中的质子转移过程。将特别关注碳酸酐酶家族，其中分子内质子转移是限速步骤。为了指导碳酸酐酶中新的、更有效的质子转移途径的实验设计，将使用基于最近提出的近似密度泛函理论（SCC-DFTB）和CHARMM力场的QM/MM方法进行一系列计算。这些计算将提供有关自由能垒、速率常数、溶剂效应、动力学同位素效应的微观信息，更重要的是各个残基对质子转移过程的贡献。在实验方面，将对选定的酶活性位点残基进行定点诱变，以引入含有人工质子穿梭的化学修饰氨基酸；这些群体的位置和性质将由计算研究指导。使用 X 射线晶体学对修饰酶进行结构测量将产生有关活性位点空间组织的有价值的信息，例如新穿梭基团的方向及其与锌离子的距离以及质子之间的水桥的配置供体和受体组。利用同位素取代和 pH 变化的动力学研究将揭示突变的催化有效性。本项目的优势在于最先进的实验和理论研究之间的连贯相互作用。实验结果有助于定义理论模拟的关键问题，而理论研究则提供了对实验结果的解释和进一步实验探索的指导。更广泛的影响：除了提供对碳酸酐酶功能的机制见解外，本项目还对理解其他酶系统中的质子转移以及揭示从头蛋白质工程的一般原理具有更广泛的影响。该研究还将为参与的学生提供充足的机会，让他们获得计算化学、有机和生物化学、分子生物学和结构生物学方面的重要技能。最后，综合理论实验研究对于激励本科生并吸引他们进行科学研究和发现具有不可估量的价值。