De Novo Mini-Metalloenyzmes with Hydrolase Activity

项目总结 拟议工作的主要目标是创建、表征和优化从头开始的双核微型 水解酶活性的金属酶。水解性裂解，也就是分子被 水的添加是一种基本的生化反应，它支配着所有生命中的细胞过程 王国。几种罕见的遗传病是由水解酶故障引起的，这使得它们对 理解。金属离子经常被用来催化这些反应，因为它们有很强的 即使在中性pH下，也能使水中的O-H键极化。一种名为DFsc的小型人工酶，它 活性部位含有两种金属离子，能够水解小分子和较大的DNA 底物。锌和钛的活性是观察到的，锌是自然界常用的，钛具有 没有已知的自然功能。这个项目试图(1)了解结构和电子因素， 有助于DFsc系统中的水解酶活性和(2)开发额外的微型金属酶 自然界未充分利用的金属，用于环境友好和催化高效的水解性裂解。 为了实现这些目标，我们将设计、制作和全面描述一系列 DFsc蛋白，其金属配位氨基酸是系统变化的。这个蛋白质文库 将使用DNA酶和磷酸酶分析以及各种金属来筛选水解酶活性 离子。成功完成这些目标将产生一个强大的模型系统，它将提供 了解天然金属水解酶和人工金属水解酶的构效关系。

PROJECT SUMMARY The major goal of the proposed work is to create, characterize, and optimize de novo binuclear mini- metalloenzymes for hydrolase activity. Hydrolytic cleavage, or the breaking apart of a molecule by the addition of water, is an essential biochemical reaction that governs cellular processes across all life kingdoms. Several rare genetic diseases come from hydrolase malfunctions, making them important to understand. Metal ions are frequently used to catalyze these reactions due to their ability to strongly polarize the O–H bond in water even at neutral pH. A small artificial enzyme called DFsc, which contains two metal ions in the active site, is capable of hydrolyzing small molecules and larger DNA substrates. Activity was observed with zinc, which is commonly used by nature, and titanium, which has no known natural function. This project seeks to (1) understand the structural and electronic factors that contribute to hydrolase activity in the DFsc system and (2) develop additional mini-metalloenzymes with metals underutilized by nature for environmentally-friendly and catalytically-efficient hydrolytic cleavage. To accomplish these goals, we will design, produce, and comprehensively characterize a series of DFsc proteins whose metal-coordinating amino acids are systematically varied. This library of proteins will be screened for hydrolase activity using DNAse and phosphatase assays and a variety of metal ions. Successful completion of these aims will result in a robust model system that will provide an understanding of the structure-activity relationships in natural and artificial metallohydrolases.