Leflunomide ring-opening by AlleyCat2 (sky blue, black) and AlleyCat2 E92Q (lavender). Experimental conditions: 25 μM enzyme, 100 mM NaCl, 25 mM HEPES pH 7.4, 0.3 mM substrate. The sample with calcium contained 10 mM CaCl2 (sky blue dots for AlleyCat2 and lavender dots for AlleyCat2 E92Q), the sample without calcium contained 50 μM EDTA (black dots for AlleyCat2).