Raymond Elizabeth A, Mack Korrie L, Yoon Jennifer H, Moroz Olesia V, Moroz Yurii S, Korendovych Ivan V
Department of Chemistry, Syracuse University, Syracuse, New York, 13244.
Protein Sci. 2015 Apr;24(4):561-70. doi: 10.1002/pro.2622. Epub 2015 Jan 13.
We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca(2+) ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue.
我们采用了一种极简主义方法来设计一种变构控制的逆羟醛酶。在非酶蛋白钙调蛋白中引入单个赖氨酸残基，使得以羟丁酮为底物的逆羟醛反应的二级速率常数提高了15000倍。由此产生的催化剂AlleyCatR活性足以在粗制细胞细菌裂解物中进行后续的定向进化。AlleyCatR的活性受Ca(2+)离子的变构调节。在没有金属离子的情况下未观察到催化作用。催化活性的提高源于底物的疏水相互作用（约2000倍）以及活性赖氨酸残基表观pKa的变化。
