Table 7. Effects of Mutating Multiple Loop Residues to Glycine 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 ± 0.6) × 10–1 | 100 ± 11 | (7.1 ± 0.2) × 103 | 51 | 0.5 | 100 |
| P39G/V40G | (8.6 ± 0.3) × 10–2 | 55 ± 12 | (1.6 ± 0.3) × 103 | 420 | 0.9 | 460 |
| P39G/V40G/S42G | (4.8 ± 0.6) × 10–2 | 55 ± 11 | (8.7 ± 0.2) × 102 | 750 | 0.9 | 830 |
| P39G/V40G/S42G/E43G | (3.9 ± 0.1) × 10–2 | 35 ± 8 | (1.1 ± 0.4) × 103 | 920 | 1.4 | 650 |
| P39G/V40G/S42G/E43G/P44G | (2.1 ± 0.5) × 10–2 | 50 ± 16 | (4.2 ± 0.4) × 102 | 1700 | 1.0 | 1700 |
| 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/V40G/S42G | (4.0 ± 0.2) × 10–2 | 34 ± 9 | (1.2 ± 0.2) × 103 | 3.8 | 1.0 | 3.6 |
| D38E/P39G/V40G/S42G/E43G/P44G | (2.3 ± 0.5) × 10–2 | 68 ± 7 | (3.4 ± 0.3) × 102 | 6.5 | 0.51 | 13 |
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.