Table 2.
Kinetic constants of the ancestral IPMDHs, their mutants, and extant IPMDHs.
| Enzyme | KmD-3-IPM (μM)a | KmNAD (μM)a | kcat (s−1)a |
|---|---|---|---|
| 25 °C | |||
| ancIPMDH-IQ | n.d.b | 45 ± 6 | 1.7 ± 0.1 |
| ancIPMDH-ML | n.d.b | 37 ± 4 | 1.2 ± 0.0 |
| ancIPMDH-IQ-VAHG | n.d.b | 77 ± 4 | 2.0 ± 0.0 |
| ancIPMDH-ML-VAHG | n.d.b | 33 ± 2 | 1.6 ± 0.0 |
| T. thermophilus IPMDH | n.d.b | 2.2 ± 0.3 | 0.37 ± 0.01 |
| B. subtilis IPMDH | n.d.b | 110 ± 10 | 2.8 ± 0.1 |
| S. cerevisiae IPMDH | n.d.b | 130 ± 10 | 5.1 ± 0.1 |
| 40 °C | |||
| ancIPMDH-IQ | 5.3 ± 0.2 | 67 ± 7 | 4.6 ± 0.1 |
| ancIPMDH-ML | 4.7 ± 0.4 | 54 ± 6 | 4.3 ± 0.1 |
| ancIPMDH-IQ-VAHG | 5.0 ± 0.2 | 230 ± 12 | 8.5 ± 0.1 |
| ancIPMDH-ML-VAHG | 5.0 ± 0.3 | 240 ± 40 | 5.0 ± 0.3 |
| T. thermophilus IPMDH | 2.3 ± 0.8 | 12 ± 1 | 2.4 ± 0.1 |
| B. subtilis IPMDH | 0.78 ± 0.11 | 420 ± 20 | 9.4 ± 0.2 |
| S. cerevisiae IPMDH | 4.5 ± 0.7 | 650 ± 70 | 20 ± 1 |
| 70 °C | |||
| ancIPMDH-IQ | 10 ± 2 | 960 ± 50 | 22 ± 0 |
| ancIPMDH-ML | 10 ± 2 | 730 ± 80 | 22 ± 1 |
| ancIPMDH-IQ-VAHG | 13 ± 3 | 4,100 ± 500 | 34 ± 2 |
| ancIPMDH-ML-VAHG | 11 ± 2 | 3,300 ± 400 | 24 ± 1 |
| T. thermophilus IPMDH | 3.7 ± 1.4 | 210 ± 10 | 79 ± 3 |
aKm and kcat were calculated from steady-state experiments performed at 25, 40, or 70 °C with an assay buffer containing 50 mM HEPES (pH 8.0), 100 mM KCl, 5 mM MgCl2, and various concentrations of D-3-IPM and NAD+. The values and standard errors were obtained by nonlinear least-square fitting of the steady-state velocity to the Michaelis–Menten equation using the Enzyme Kinetics module of SigmaPlot version 13.0, from Systat Software, Inc., San Jose California USA, www.systatsoftware.com.
bKm values for D-3-IPM at 25 °C could not be determined because appropriately designed steady-state kinetic experiments would include reactions that use very small amounts of D-3-IPM, which would be consumed quickly and preclude accurate velocity measurements.