How directed evolution reshapes the energy landscape in an enzyme to boost catalysis.

The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of NMR, crystallographic, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and achieve a nearly billionfold rate acceleration. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-state stabilization. Explicit sampling of conformational sub-states during design, and specifically stabilizing productive over all unproductive conformations, could speed up the development of protein catalysts for many chemical transformations.