Table 3. Effects of Mutation of Met112 and Ala114 on KSI Kinetic Parameters with an Asp (WT) and Glu (D38E) General Basea.
| enzyme | kcat (s–1) | KM (μM) | kcat/KM (M–1 s–1) | kcat ratio (WT/mutant)b | KM ratio (WT/mutant)b | kcat/KM ratio (WT/mutant)b |
|---|---|---|---|---|---|---|
| WT | 36 ± 2 | 50 ± 4 | (7.2 ± 0.3) × 105 | (1) | (1) | (1) |
| M112A | 1.1 ± 0.1 | 28 ± 6 | (3.9 ± 0.5) × 104 | 33 | 1.8 | 18 |
| A114G | (1.6 ± 0.2) × 10–1 | 35 ± 4 | (4.6 ± 0.3) × 103 | 225 | 1.4 | 160 |
| enzyme | kcat (s–1) | KM (μM) | kcat/KM (M–1 s–1) | kcat ratio (D38E/mutant)c | KM ratio (D38E/mutant)c | kcat/KM ratio (D38E/mutant)c |
|---|---|---|---|---|---|---|
| D38E | (1.5 ± 0.2) × 10–1 | 35 ± 3 | (4.3 ± 0.1) × 103 | (1) | (1) | (1) |
| D38E/M112A | (1.5 ± 0.1) × 10–2 | 20 ± 4 | (7.5 ± 0.3) × 102 | 10 | 1.8 | 5.7 |
| D38E/A114G | (1.0 ± 0.2) × 10–2 | 19 ± 2 | (5.3 ± 0.2) × 102 | 15 | 1.8 | 8.1 |
a
Kinetics experiments were performed for the 5(10)-EST substrate for which the chemical steps of the KSI reaction are rate-limiting.4,58
b
By definition, the ratio for WT KSI is 1, as represented by the values in parentheses.
c
By definition, the ratio for D38E KSI is 1, as represented by the values in parentheses.