One obvious feature in the FuncLib results is the frequent prediction among the highly scored variants of a phenylalanine residue at position 260 (vs. the Leu residue present in the background "WT" protein, denoted here as GNCA4-WT). This is interesting, because, although close to the de novo active site, position 260 belongs to a β-strand and its side chain is actually opposite the active site. Therefore, as a first step to explore the FuncLib predictions we assessed the effect of a single L260F mutation on Kemp elimination catalysis. We observe that this L260F mutation by itself is able to enhance both the catalytic efficiency and turnover number by about 2-fold. While this is only a moderate increase in activity, it is already comparable to those for the single improved variant obtained from the screening of a non-focused, random mutation library (Table ).