The reactions were quenched using 50 μL of 2.5 M NaOH, and the amount of the product pnitrophenol was assayed from the absorption at 400 nm using the molar extinction coefficient of 18,300 M -1 cm -1 . Reaction blanks were made using identical conditions replacing the enzyme with buffer to correct for non-enzymatic hydrolysis of the substrate. The amount of product released and elapsed time were used to calculate initial rates. These data were fitted to the Michaelis-Menten equation to obtain steady-state kinetic parameters. Kinetic data were obtained on both variants as a function of pH to obtain pH-rate profiles which were fitted to Eqs. 1 and 2. In Eq. 2 KS2 was set to the second ionization constant of the substrate pNPP (pKa = 4.96). These equations relate the dependence of the observed values of kcat, or kcat/KM, to their maximal, or limiting, values as a function of pH, where catalysis is dependent on two ionizable enzymatic residues, one protonated and the other deprotonated.