The 18 O KIE in the scissile P-O bond and the 15 N KIE in the leaving group were measured using the competitive method and isotope ratio mass spectrometry. Figure shows the positions in the substrate where KIEs were measured and the isotopic isomers used. KIEs measured by the competitive method are isotope effects on V/K, the part of the mechanism up to the first irreversible step, which is the first step shown in Figure , cleavage of the pNPP substrate. Concentrated protein aliquots were thawed on ice and diluted to 9.26 and 17.45 µM for Chimera 3 and Chimera 4 respectively in reaction with the buffer base mix. Natural abundance pNPP was used for measurements of 15 (V/K). N, O-labeled pNPP used for measurement of O isotope effects by the remote label method were synthesized using previously published methods. Isotope effect determinations were carried out in triplicate, at 25 °C in 100 mM Bis-tris at pH 5.5, by adding 200 µL enzyme and 108 micromoles pNPP in 5 mL of buffer and allowed to react until approximately 50% completion, approximately 2-3 hours. The enzymatic reactions were then stopped by titration to pH 4 with HCl, and the p-nitrophenol product extracted using diethyl ether and purified by sublimation. The residual substrate in the aqueous layer was completely hydrolyzed using bovine alkaline phosphatase at pH 9, and the p-nitrophenol released was isolated in the same way. The KIEs were calculated from the nitrogen isotopic ratios in the p-nitrophenol product at partial reaction (Rp), in the residual substrate (Rs), and the starting reactant (Ro). Eq. 3 was used to calculate the observed KIE from Rs and Ro at fraction of reaction f, and Eq. 4 from Rp and Ro. These were the same within experimental error and averaged to give the final results.