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

这一合作项目涉及一种理论和实验相结合的方法，旨在更好地了解酶中的质子转移过程。一个具体的重点将放在碳酸酐酶家族上，其中分子内质子转移是速率限制步骤。为了指导碳酸氢酶中新的、更有效的质子转移途径的实验设计，基于最近提出的近似密度泛函理论(SCC-DFTB)和CHARMM力场，将用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，将对酶的选定活性部位残基进行定点突变，以引入含有人造质子航天飞机的化学修饰氨基酸；这些基团的位置和性质将通过计算研究来指导。利用X射线结晶学对修饰酶的结构进行测量，将获得有关活性中心的空间组织的有价值的信息，如新的穿梭基团的取向及其与锌离子的距离，以及质子给体和受体基团之间的水桥构型。动力学研究，利用同位素替代和pH变化，将揭示突变的催化效果(S)。本项目的优势在于最先进的实验和理论研究之间连贯的相互作用。实验结果有助于确定理论模拟的关键问题，而理论研究反过来又提供了对实验结果的解释和对进一步实验探索的指导。广泛的影响：除了提供对碳酸酐酶功能的机制洞察之外，本项目还对理解其他酶系统中的质子转移和揭示从头蛋白质工程的一般原理有更广泛的影响。这项研究还将为参与的学生提供充分的机会，以获得计算化学、有机化学和生物化学、分子生物学和结构生物学方面的重要技能。最后，理论和实验相结合的研究对于激励本科生和吸引他们从事科学研究和发现将是非常有价值的。

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.