TABLE 2.
Nucleotide binding parameters of NBD1-M(141–534) and NBD2(520–854) in the presence or absence of GdmCl
| NBD1-M WT |
NBD1-M E209A |
NBD2 WT,a no GdmCl | |||
|---|---|---|---|---|---|
| No GdmCl | 10 mm GdmCl | No GdmCl | 10 mm GdmCl | ||
| MANT-ADPb | |||||
| kon (μm−1 s−1) | 0.12 | 0.15 | NDc | ND | ND |
| koff (s−1) | 4.0 | 1.2 | ND | ND | ND |
| KD (μm) | 33 | 8 | ND | ND | ND |
| MANT-dADPb | |||||
| kon (μm−1 s−1) | 0.34 | 0.36 | 1.00 | 1.09 | 4.2 |
| koff (s−1) | 3.3 | 2.8 | 4.1 | 4.2 | 0.015 |
| KD (μm) | 10 (5)d | 8 | 4 | 4 | 0.004 |
| Unlabeled nucleotides | |||||
| KD(ADP) (μm) | 13.7 (6.7)d | 2.4 | 6.5 | 6.9 | 0.5 |
| KD(ATP) (μm) | 1520 (510)d | 122 | 1190 | 1320 | 89 |
a Nucleotide binding parameters of NBD2(520–854) as published in Ref. 31.
b The differences in kon between MANT-ADP and MANT-dADP binding in the absence of GdmCl can be explained when taking into consideration that only one of the two different MANT-ADP isomers binds efficiently, and its concentration in the mixture is lower than the overall concentration of MANT-ADP.
c ND, not determined.
d Values in parentheses refer to measurements at higher protein concentration of [NBD1-M] = 20 μm, whereas all other binding parameters were determined at low protein concentration of [NBD1-M] = 2 μm.