Determination of Kemp elimination activity were carried out at 25 ºC HEPES 10 mM or 10 mM sodium phosphate (in all cases with 100 mM NaCl), depending on the pH range, as has been previously described in ref. . Experiments were routinely carried out in the presence of acetonitrile to increase the solubility of the substrate and expand its experimental concentration range, thus facilitating the detection of curvature in Michaelis plots and, therefore, the reliable determination of turnover numbers. 5% acetonitrile was used in most cases, although experiments with higher and lower acetonitrile contents were also performed (see the Results and Discussion for details). It is to be noted that, even in those cases in which no acetonitrile is added on purpose, a small amount of the cosolvent is present because the stock solution of the substrate is prepared in acetonitrile. The approximate substrate ranges used depend on acetonitrile concentration, reflecting the substrate solubility (Table ).