Using a BioTek plate reader (Winooski, VT), kinetic assays were performed by measuring the absorbance of the paraoxon leaving group at 405 nm over 10 min of reaction time. Various amounts of paraoxon (final concentration range: 0.001 -3.0 mM) were added to 193.7 L of buffer containing 100 mM CHES and 75 M of Zn(OAc) 2 , pH = 9.0, reactions were started by adding 2 L of WT or mutant OPH. The changes of absorbance at 405 nm over time were used to calculate initial velocities as a function of the various substrate concentrations. Then the initial velocity data, along with corresponding paraoxon concentrations, were plotted and analyzed by the Michaelis-Menten equation to obtain V max and K M using GraphPad (GraphPad Software, Inc., La Jolla, CA, USA; Swift 1997). The catalytic rate (k cat ) was calculated by the ratio of maximum velocity (V max ) and mutant concentration. Each kinetic measurement condition was performed in triplicate and standard deviation was calculated using GraphPad.