Table 4. Comparison of k_cat/K_M Values for Isomerization of Protiated 5(10)-EST in H₂O and 4,4-Dideuterated 5(10)-EST in D₂O by Semisynthetic KSIs^a.

residue at position 16	kcat/KM for protiated 5(10)-EST in H2O (M–1 s–1)	kcat/KM for 4,4-dideuterated 5(10)-EST in D2O (M–1 s–1)	ratio of kcat/KM values for protiated and 4,4-dideuterated 5(10)-EST
Tyr (WT)	(8.2 ± 1.6) × 104	(1.2 ± 0.3) × 104	6.8 ± 2.1
2-F-Tyr	(8.3 ± 0.3) × 104	(1.3 ± 0.2) × 104	6.3 ± 1.0
3-F-Tyr	(6.4 ± 1.2) × 104	(7.4 ± 1.2) × 103	8.7 ± 2.2
2,6-F2-Tyr	(6.5 ± 0.8) × 104	(9.5 ± 1.6) × 103	6.8 ± 1.4

^aConditions: 40 mM potassium phosphate, 1 mM EDTA, 2% DMSO (v/v), pH 7.2 (H₂O) or pD 7.6 (D₂O). Values of k_cat/K_M are from best fits to data in Figure S13 (Supporting Information) and are shown together with standard deviations from 3 to 4 independent experiments using enzyme concentrations varied over a 3-fold range, using either 6 or 12 μM substrate. Uncertainties in the ratios of k_cat/K_M values were obtained by error propagation.