Table 5. Comparison of kcat Values for Isomerization of Protiated 5(10)-EST in H2O and 4,4-Dideuterated 5(10)-EST in D2O by Semisynthetic KSIsa.
| residue at position 16 | kcat for protiated 5(10)-EST (in H2O) (s–1) | kcat for 4,4-dideuterated 5(10)-EST (in D2O) (s–1) | ratio of kcat values |
|---|---|---|---|
| Tyr (WT) | 3.2 ± 0.5 | (3.9 ± 0.7) × 10–1 | 8.1 ± 2.0 |
| 2-F-Tyr | 3.1 ± 0.4 | (4.0 ± 0.4) × 10–1 | 7.7 ± 1.2 |
| 3-F-Tyr | 2.9 ± 0.6 | (3.5 ± 0.5) × 10–1 | 8.2 ± 2.0 |
| 2,6-F2-Tyr | 3.4 ± 0.3 | (3.9 ± 0.1) × 10–1 | 8.9 ± 0.8 |
a
Conditions: 40 mM potassium phosphate, 1 mM EDTA, 2% DMSO (v/v), pH 7.2 (H2O) or pD 7.6 (D2O). Values of kcat are from best fits to data in Figure S14 (Supporting Information) collected at 300 μM substrate, a concentration shown to be saturating in independent experiments (Figure S5, Table S3, Supporting Information). Values are shown together with standard deviations from 2 to 3 independent experiments using enzyme concentrations varied over a 4-fold range. Uncertainties in the ratios of kcat values were obtained by error propagation.