|
Kinetic parameters
|
---|
Enzyme
|
Variable substrate
|
k
cat
(s-1)
|
Km(μM)
|
k
cat
/Km(mM-1s-1)
|
---|
|
BAL
| | | |
Wild type
| |
3.36 ± 0.13
|
98 ± 15
|
35 ± 4
|
A441C
| |
2.99 ± 0.19
|
90 ± 6
|
33 ± 2
|
A441S
| |
3.29 ± 0.12
|
119 ± 8
|
28 ± 3
|
A441T
| |
2.64 ± 0.16
|
180 ± 6
|
15 ± 1
|
A441V
| |
0.54 ± 0.05
|
512 ± 79
|
1.1 ± 0.3
|
A441F
| |
0.29 ± 0.02
|
605 ± 26
|
0.48 ± 0.05
|
A441I
| |
0.74 ± 0.05
|
1791 ± 115
|
0.41 ± 0.05
|
|
NAD
+
| | | |
Wild type
| |
4.25 ± 0.16
|
22 ± 2
|
195 ± 8
|
A441C
| |
2.20 ± 0.09
|
14 ± 1
|
179 ± 17
|
A441S
| |
3.27 ± 0.18
|
29 ± 3
|
114 ± 20
|
A441T
| |
2.39 ± 0.00
|
24 ± 4
|
100 ± 15
|
A441V
| |
0.51 ± 0.00
|
6.4 ± 0.5
|
80 ± 5
|
A441F
| |
0.39 ± 0.02
|
18 ± 1
|
22 ± 0
|
A441I
| |
0.68 ± 0.03
|
2.8 ± 0.0
|
243 ± 11
|
- Initial velocities were obtained at 30°C in 50 mM HEPES-KOH buffer, pH 8.0, containing 0.1 mM EDTA. In the experiments with variable BAL, the fixed concentration of NAD+ was 0.2 mM, and in the experiments with variable NAD+ the fixed BAL concentrations were at least 10-times their appKm values estimated for each enzyme at fixed 0.2 mM NAD+. The apparent kinetic parameters were estimated by non-linear regression fit of the experimental data to the Michaelis-Menten equation. The values given in the Table are the mean ± standard deviation of the kinetic parameters estimated in two duplicate saturation experiments performed with enzymes from two different purification batches. Values for k
cat
are expressed per subunit.