Increasing acetonitrile concentrations somewhat depresses the catalytic activity. Two factors may contribute to this. First, since acetonitrile increases substrate solubility, it is also stabilizing the free (non-bound) substrate and thus potentially increasing some of the relevant kinetic free energy barriers. In addition, the interaction of acetonitrile molecules with the protein may directly modify such barriers, through small alterations in the structure or dynamics. The conjunction of these two factors could perhaps be behind the somewhat complex dependency seen for the catalytic efficiency of the GNCA-12 variant (left panel, Figure ). In any case, these speculative interpretations do not affect the main point of Figure , namely that the extrapolations to zero acetonitrile concentration are rather short (even for kcat) and, therefore, there is little doubt about the reliability of the extrapolated values. The short extrapolation leads to a catalytic efficiency and a turnover number of about 2•10 4 M -1 •s -1 and 10 2 s -1 . These values are well within the ranges of catalytic parameters for modern natural enzymes and, in particular, the value 10 2 s -1 for kcat is about one order of magnitude higher than the median value of the kcat distribution for modern enzymes. Finally, we have used X-ray crystallography to determine the 3D-structures of the catalytically optimized GNCA4-2, GNCA4-12 and GNCA4-19 variants, the first of which has a transition state analog bound at the de novo active site. These particular structures were chosen as they are all highly active variants, in terms of the measured rates within the available substrate concentration range (Figure ), with improved catalytic parameters over GNCA4-WT (Table and Figure ).