Department of Pharmaceutical Chemistry, University of California-San Francisco, San Francisco, CA 94158-2550, USA.
Proc Natl Acad Sci U S A. 2012 Oct 2;109(40):16179-83. doi: 10.1073/pnas.1208076109. Epub 2012 Sep 17.
Synthetic cavitands and protein cavities have been widely studied as models for ligand recognition. Here we investigate the Met102 → His substitution in the artificial L99A cavity in T4 lysozyme as a Kemp eliminase. The resulting enzyme had k(cat)/K(M) = 0.43 M(-1) s(-1) and a (k(cat)/K(M))/k(uncat) = 10(7) at pH 5.0. The crystal structure of this enzyme was determined at 1.30 Å, as were the structures of four complexes of substrate and product analogs. The absence of ordered waters or hydrogen bonding interactions, and the presence of a common catalytic base (His102) in an otherwise hydrophobic, buried cavity, facilitated detailed analysis of the reaction mechanism and its optimization. Subsequent substitutions increased eliminase activity by an additional four-fold. As activity-enhancing substitutions were engineered into the cavity, protein stability decreased, consistent with the stability-function trade-off hypothesis. This and related model cavities may provide templates for studying protein design principles in radically simplified environments.
人工 L99A 空腔中的 T4 溶菌酶 Met102→His 取代作为 Kemp 消除酶被广泛研究。该酶的 k(cat)/K(M) = 0.43 M(-1) s(-1)，(k(cat)/K(M))/k(uncat) = 10(7)在 pH 5.0 下。该酶的晶体结构在 1.30 Å 处确定，以及四个底物和产物类似物复合物的结构。缺乏有序的水或氢键相互作用，以及常见的催化碱（His102）存在于其他疏水、埋藏的空腔中，促进了反应机制及其优化的详细分析。随后的取代使消除酶的活性增加了四倍。随着活性增强取代被引入空腔，蛋白质稳定性降低，这与稳定性-功能权衡假说一致。这些和相关的模型空腔可以为在简化环境中研究蛋白质设计原则提供模板。
