1. Although many of the variants display diminished activity compared with the background V4 variant, several variants are significantly more active (Figure ). In particular, variant V4-4 shows, at pH 8.5, a catalytic efficiency, kcat/KM, of (4.3±0.1)•10 5 M -1 s -1 , and a catalytic rate constant, kcat, of 1700±230 s -1 . We emphasize that this is actually a very high kcat value that is above most reported kcat values for engineered/evolved de novo enzymes and modern natural enzymes. 2. The activity range across the designed variants (which spans impaired and improved activity) is ~50-fold. It is important for design purposes to emphasize that this ~50-fold change in activity is not achieved through active site redesign or through impairment of the catalytic machinery. Rather, it is achieved through mutating side-chains that are outside the active-site, and this difference is significant. Certainly, non-active-site mutations could impair activity if they are disruptive and prevent proper folding to the functional 3D-functional structure. However, our combinations of mutations are not disruptive: they are stabilizing, in fact, as shown in Figure . Therefore, the reported 50-fold modulation in rate supports that large effects on activity may be achieved by targeting distant hot spots, which is an important and non-intuitive result. The overall activity enhancement with respect to the background variant used in our study is smaller, at ~3-fold, but this only reflects our choice of selecting as background an already highly active enzyme that had been previously extensively optimized (larger enhancements could have likely been obtained with a less optimized background). kcat/KM of 4.3 x 10 5 M -1 s -1 and it is not impaired in terms of stability. Rather, its denaturation temperature is a few degrees higher than that of the highly stable background used.