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

该合作项目采用理论与实验相结合的方法，旨在更好地理解酶中的质子转移过程。一个特定的重点将放在碳酸酐酶家族，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和更有效的质子转移途径的实验设计，将基于最近提出的近似密度泛函理论（SCC-DFTB）和CHARMM力场，使用QM/MM方法进行一系列计算。这些计算将提供关于自由能垒、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，单个残基对质子转移过程的贡献。在实验方面，将对酶的选定活性位点残基进行定点诱变，以引入含有人工质子穿梭体的化学修饰氨基酸；这些群体的位置和性质将以计算研究为指导。利用x射线晶体学对修饰酶进行结构测量，将获得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到锌离子的距离，以及质子供体和受体基团之间水桥的配置。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。实验结果有助于确定理论模拟的关键问题，而反过来，理论研究既提供了对实验结果的解释，又为进一步的实验探索提供了指导。更广泛的影响：除了提供对碳酸酐酶功能的机制见解外，本项目对理解其他酶系统中的质子转移以及揭示从头开始的蛋白质工程的一般原理具有更广泛的影响。本研究也将为参与的学生提供充分的机会，以获得计算化学、有机与生物化学、分子生物学和结构生物学的重要技能。最后，理论与实验相结合的研究对于激励和吸引本科生从事科学研究和发现具有不可估量的价值。

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.