Table 1. Kinetic Parameters for Wild-Type and Mutant hlGPDH-Catalyzed Reactions of Whole Substrate DHAP and Truncated Substrate Glycolaldehyde at pH 7.5 (triethanolamine buffer) and an Ionic Strength of 0.12 (NaCl)a.
| enzyme | kcat (s–1)b | Km (M)b | kcat/Km (M–1 s–1)b | (kcat/Km)E (M–1 s–1)c |
|---|---|---|---|---|
| WT,d 10.8 kcal/mole | 240 ± 10 | (5.2 ± 0.3) × 10–5 | (4.6 ± 0.3) × 106 | (5.0 ± 0.6) × 10–2 |
| R269A,d 9.1 kcal/molf | (5.9 ± 0.4) × 10–3 | (5.7 ± 0.5) × 10–3 | 1.0 ± 0.1 | ≤0.003g |
| N270A, 3.1 kcal/mole 5.6 kcal/molf | 9.0 ± 0.5 | (2.5 ± 0.2) × 10–2 | 360 ± 35 | 2.0 ± 0.2 |
| R269A/N270A, 11.5 kcal/molf | (2.8 ± 0.1) × 10–4 | (1.5 ± 0.1) × 10–2 | (1.7 ± 0.1) × 10–2 | ≤0.003g |
a
The uncertainty in the kinetic parameters is the standard error from least-squares fits of the kinetic data.
b
Kinetic parameter for hlGPDH-catalyzed reactions at saturating [NADH], calculated for the carbonyl form of DHAP.15
c
Calculated for the carbonyl form of GA at saturating [NADH].15
d
From ref (17).
e
The intrinsic dianion binding energy, calculated from the ratio of kcat/Km for hlGPDH-catalyzed reduction of DHAP and GA.
f
Effect of the mutation on the activation barrier ΔG⧧ for wild-type hlGPDH (Scheme 1), determined as described in the text.
g
Estimated upper limit for (kcat/Km)E, calculated as described in the Supporting Information.