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 Ca2+ 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.
|Original language||English (US)|
|Number of pages||10|
|State||Published - Apr 1 2015|
- enzyme catalysis
- protein design
ASJC Scopus subject areas
- Molecular Biology