Table 1. Kinetic parameters for wild-type enzyme and mutant proteins for half reaction with HCO3- varieda.
| Vmaxb (μmol min-1mg-1) | Kmb(mM) | Km (fold change) | Kcatc (min-1) | Kcat/Km (μM-1 min-1) | Kcat/Km (fold change) | |
|---|---|---|---|---|---|---|
| wild-type | 0.043 ± 0.002 | 3.9 ± 0.5 | 0 | 3.8 ± 0.2 | 1 ± 0.4 | 1 |
| E73A | 0.17 ± 0.01 | 33 ± 6 | 9 | 15 ± 1 | 0.5 ± 0.2 | 2 |
| Y152A | 0.03 ± 0.0007 | 5 ± 0.3 | 1 | 1.9 ± 0.04 | 0.4 ± 0.1 | 3 |
| Y152F | 0.05 ± 0.001 | 2.8 ± 0.02 | 1 | 3.2 ± 0.08 | 1 ± 0.4 | 1 |
| D153A | 0.15 ± 0.01 | 15 ± 4 | 4 | 14 ± 1 | 0.9 ± 0.3 | 1 |
| R155A | 0.10 ± .003 | 7 ± 0.7 | 2 | 8.6 ± 0.3 | 1 ± 0.4 | 1 |
| R271A | Inactived | nd | nd | nd | nd | nd |
| R271Q | Inactived | nd | nd | nd | nd | nd |
| R271K | 0.0009 ± 0.00008 | 80 ± 20 | 22 | 0.08 ± 0.007 | 0.001 ± 0.0004 | 1,000 |
| H273A | Inactived | nd | nd | nd | nd | nd |
| H273Q | Inactived | nd | nd | nd | nd | nd |
| K353A | Inactived | nd | nd | nd | nd | nd |
| K353R | Inactived | nd | nd | nd | nd | nd |
a
Reported data are average of duplicates and errors are those obtained from curve fitting (equation 1) the appropriate data set.
b
Steadystate values were determined using the ATP coupled assay system without AIR, with the concentration of ATP fixed and the concentration of bicarbonate is varied. Km values are derived from the total amount of bicarbonate in solution (including background and added bicarbonate).
c
Calculated using the molecular weight of the dimer.
d
No measurable activity up to a protein concentration of 700- 4500 μg/mL.