Table 4. Effects of Individual Mutations of Catalytic Base Loop Residues 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) |
| P39G | (7.1 ± 0.2) × 10–1 | 100 ± 2 | (7.1 ± 0.2) × 103 | 51 | 0.5 | 100 |
| V40G | (5.1 ± 0.2) × 10–1 | 40 ± 2 | (1.3 ± 0.3) × 104 | 71 | 1.3 | 57 |
| G41A | 2.6 ± 0.2 | 50 ± 3 | (5.2 ± 0.2) × 104 | 14 | 1.0 | 14 |
| G41V | 1.8 ± 0.4 | 40 ± 3 | (4.5 ± 0.4) × 104 | 20 | 1.3 | 16 |
| S42G | 6.3 ± 0.2 | 34 ± 3 | (1.9 ± 0.4) × 105 | 5.7 | 1.5 | 3.9 |
| E43G | 16 ± 0.2 | 25 ± 4 | (6.4 ± 0.4) × 105 | 2.3 | 2.0 | 1.1 |
| P44G | 3.4 ± 0.3 | 50 ± 5 | (6.8 ± 0.2) × 104 | 11 | 1.0 | 11 |
| 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 ± 13 | (4.3 ± 0.1) × 103 | (1) | (1) | (1) |
| D38E/P39G | (1.2 ± 0.5) × 10–2 | 30 ± 9 | (4.0 ± 0.1) × 102 | 13 | 1.2 | 11 |
| D38E/V40G | (4.9 ± 0.3) × 10–2 | 25 ± 9 | (2.0 ± 0.2) × 103 | 3.1 | 1.4 | 2.2 |
| D38E/G41V | (4.0 ± 0.2) × 10–2 | 35 ± 5 | (1.1 ± 0.4) × 103 | 3.8 | 1.0 | 3.8 |
| D38E/S42G | (1.1 ± 0.2) × 10–1 | 28 ± 9 | (3.9 ± 0.1) × 103 | 1.4 | 1.3 | 1.1 |
| D38E/E43G | (4.0 ± 0.2) × 10–2 | 50 ± 10 | (8.0 ± 0.1) × 102 | 3.8 | 0.7 | 5.4 |
| D38E/P44G | (5.0 ± 0.1) × 10–2 | 62 ± 4 | (8.1 ± 0.1) × 102 | 3.0 | 0.6 | 5.3 |
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.