Table 1. Comparative Kinetic Parameters of the Single-Trp Mutants Following Insertion of Y25A and V260A, 30 °C.
|
KM, mM |
Dkcat/D(kcat/KM)b |
||||||
|---|---|---|---|---|---|---|---|
| mutant | kcat, s–1 | NAD+ | h-BnOH | Dkcata | BnOH | NAD+ | ΔΔH⧧, kcal/molc |
| WTd | 8.0 (1.0) | 1.1 (0.1) | 6.8 (0.5) | 3.1 (0.2) | 0.8 (0.2) | 1.0 (0.1) | 6.7 (0.9) |
| Y25Ae | 5.1(0.5) | 1.0 (0.5) | 16.5 (2.1) | 2.7 (0.1) | 0.4 (0.1) | 1.0 (0.3) | N.A.h |
| V260Af | 1.9 (0.2) | 10.0 (1.6) | 4.2 (0.8) | 4.1 (0.7) | 1.0 (0.1) | 1.0 (0.2) | 18.7 (1.6) |
| W87ing | 3.4 (0.2) | 1.8 (0.2) | 3.1 (0.4) | 2.5 (0.1) | 1.0 (0.2) | 1.0 (0.2) | 5.4 (0.4) |
| W87in:Y25A | 2.5 (0.3) | 0.5 (0.1) | 0.8 (0.1) | 3.5 (0.3) | 0.5 (0.1) | 1.0 (0.1) | N.A.h |
| W87in:V260A | 1.4 (0.2) | 8.3 (1.1) | 8.2 (1.6) | 4.2 (0.2) | 1.0 (0.1) | 1.0 (0.1) | 15.2 (1.2) |
| W87in:Y25A:V260A | 1.9 (0.2) | 9.7 (1.5) | 5.4 (1.3) | 3.5 (0.6) | 1.1 (0.4) | 1.4 (0.5) | 17.8 (1.3) |
| W167ing | 3.0 (0.1) | 1.3 (0.3) | 6.0 (0.2) | 2.2 (0.1) | 0.9 (0.1) | 0.8 (0.1) | 5.7 (0.4) |
| W167in:Y25A | 6.3 (0.3) | 0.9 (0.2) | 10.9 (2.0) | 1.8 (0.1) | 0.4 (0.1) | 0.3 (0.1) | N.A.h |
| W167in:V260A | 1.0 (0.1) | 10.2 (0.8) | 12.9 (1.5) | 2.8 (0.3) | 1.0 (0.1) | 0.9 (0.2) | 12.2 (1.3) |
| W167in:Y25A:V260A | 2.5 (0.1) | 14.8 (0.6) | 10.1 (0.7) | 3.5 (0.5) | 0.9 (0.2) | 0.9 (0.2) | 14.5 (2.5) |
a
Computed as kcat(h-BnOH)/kcat(d-BnOH).
b
kcat /KM for substrate and cofactor are extracted from fits of the individual trials of h-BnOH or d-BnOH.
c
Computed as the difference of ΔH⧧ <30 °C and ΔH⧧ >30 °C.
d
From ref (18).
e
From ref (24).
f
From ref (17).
g
From ref (26).
h
N.A., not available, due to a lack of a break in the Arrhenius plots. ΔH⧧ = 12.1 (0.5) kcal/mol for Y25A24 and 13.9 (0.5) and 13.8 (0.5) kcal/mol for W87in:Y25A and W167in:Y25A, respectively.