Enzyme-like proteins by computational design
Enzyme-like proteins by computational design
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DOI：
10.1073/pnas.251555398
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发表时间：
2001-12-04
影响因子：
11.1
通讯作者：
Mayo, SL
中科院分区：
文献类型：
--
作者：
Bolon, DN;Mayo, SL
我们报告了一种旨在产生酶样蛋白质催化剂的计算设计程序的开发和初步实验验证，称为“原酶”。我们的设计方法采用“计算和构建”策略，该策略基于控制蛋白质稳定性和催化机制的物理/化学原理。通过使用催化惰性的108残基大肠杆菌硫氧还蛋白作为支架，组氨酸介导的对硝基苯醋酸酯的亲核水解作为模型反应，并使用ORBIT蛋白质设计软件计算序列，活性位点扫描确定了底物结合所需的两个有希望的催化位置和周围的活性位点突变。实验表明，这两种候选原酶的催化活性显著高于背景。其中一种蛋白质PZD2在高底物浓度下表现出“爆发”相动力学，与稳定的酶中间体的形成一致。PZD2的动力学参数与早期的催化抗体相当。但与催化Ab设计不同的是，我们的设计过程与折叠无关，这为研究蛋白质折叠与蛋白质功能可进化性之间的关系提供了一种可能的机制。
We report the development and initial experimental validation of a computational design procedure aimed at generating enzymelike protein catalysts called "protozymes." Our design approach utilizes a "compute and build" strategy that is based on the physical/chemical principles governing protein stability and catalytic mechanism. By using the catalytically inert 108-residue Escherichia coli thioredoxin as a scaffold, the histidine-mediated nucleophilic hydrolysis of p-nitrophenyl acetate as a model reaction, and the ORBIT protein design software to compute sequences, an active site scan identified two promising catalytic positions and surrounding active-site mutations required for substrate binding. Experimentally, both candidate protozymes demonstrated catalytic activity significantly above background. One of the proteins, PZD2, displayed "burst" phase kinetics at high substrate concentrations, consistent with the formation of a stable enzyme intermediate. The kinetic parameters of PZD2 are comparable to early catalytic Abs. But, unlike catalytic Ab design, our design procedure is independent of fold, suggesting a possible mechanism for examining the relationships between protein fold and the evolvability of protein function.