Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States.
J Am Chem Soc. 2012 Dec 19;134(50):20513-20. doi: 10.1021/ja3094795. Epub 2012 Dec 5.
The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.
理性修饰酶以执行新颖的化学转化的能力对于快速生产下一代蛋白质治疗药物至关重要。在这里，我们描述了使用化学原理来鉴定一种天然存在的酸活性肽酶，以及随后使用计算蛋白质设计工具来重新设计其对存在于麸质中的免疫原性成分的特异性，而这些成分被认为是引起乳糜泻的原因。该工程酶的 k(cat)/K(M) 值为 568 M(-1) s(-1)，比天然模板酶对模型谷氨酰胺四肽的蛋白水解活性高 116 倍，对谷氨酰胺肽的免疫原性部分的底物特异性提高了 800 多倍。该计算工程酶能够抵抗消化蛋白酶的蛋白水解作用，并在不到一个小时的时间内降解与乳糜泻相关的 95%以上的免疫肽。因此，通过鉴定具有与最终目标相关的固有品质的天然酶，并使用计算建模重新定义其底物特异性，我们能够生成一种具有乳糜泻治疗潜力的酶。
