“Eliminating the Unknown: The Artificial Evolution of Kemp Eliminase”
The Kemp Elimination reaction1, is a non-natural reaction that has been used as a target for the design of enzymes for the past 20 years2. However, most of designed Kemp eliminases presented low catalytic efficiency. A dramatic acceleration on efficiency was obtained when the computationally designed Kemp eliminase HG 3 (kcat = 0.68 s-1) was subjected to 17 rounds of mutations, generating the version HG 3.17 ( kcat = 700 s-1)3. Despite the 1000-fold acceleration, Kemp HG 3.17 is inactivated at temperatures far below its melting temperature. We investigated the origin of the inactivation process by observing the enzyme behavior using different techniques (activity assays, thermofluor, X-ray crystallography and NMR). Our results suggest that at higher temperatures Kemp HG 3.17 adopts an inactive conformational that hinders substrate binding.