The enhancements in catalytic activity reported above have been obtained by following a procedure that did not explicitly take the structure or stabilization of the transition state into account. That is, we simply focused our screening to regions of the sequence space that are meaningful (positions near and at the active site) and also safe to mutate, in the sense that the predicted multiple-mutation variants are not stability-impaired and their folding is not compromised. We were then interested in exploring the extent to which computational calculations on the catalytic step itself could be used to further focus and guide the screening. To this end, we have used the empirical valence bond (EVB) approach to probe the catalytic activity of the FuncLib predictions, as this approach has been extensively used to successfully study enzyme catalysis in general, and Kemp elimination in particular. In particular, this allows us to build on our recent work, in which used the EVB approach to study the evolution of multiple active site configurations in the de novo designed Kemp eliminase, KE07. In the present work, we follow the protocol presented in ref. , as described in brief in the Materials and Methods.