Table 1.
Functional and Molecular Parameters for EGK A65T and R369A Variants1
| Enzyme | EGK | R369A EGK | A65T EGK |
|---|---|---|---|
| IIAGlc inhibition | |||
| K0.5, µM | 6.3 (5.3, 7.4) | 2.5 (1.3, 3.8) | 19 (10, 28) |
| W | 0.07 (0.03, 0.12) | 0.38 (0.32, 0.43) | 0.13 (0, 0.28) |
| MgATP-IIAGlc allosteric coupling |
|||
| Vo, mM s−1 | 19.3 (18.8, 19.8) | 1.9 (1.8, 2) | 22.8 (22.1, 23) |
| KATPo, µM | 12 (11.6, 14) | 230 (175, 280) | 14 (13,16) |
| KIIo, µM | 7 (5, 10) | 2 (0.7,3.4) | 28 (14, 41) |
| KII∞, µM | 4.6 (3.3, 5.9) | 1.8 (0.3, 3.3) | 8 (2, 13) |
| Q | 1.5 (0.9, 2.2) | 1.1 (0.6, 1.6) | 3.6 (1.9, 5.3) |
| W | 0.03 (0.01, 0.05) | 0.53 (0.45, 0.6) | 0.18 (0.15, 0.21) |
| Initial-velocity2 | |||
| KATP, µM | 9 (8, 11) | 153 (104, 201) | 15 (11,19) |
| KiATP, µM | 54 (31, 76) | 157 (107, 206) | 34 (17, 51) |
| Kgol, µM | 5 (3, 7) | 40 (33, 46) | 15 (11, 19) |
| Kigol, µM | 30 (18, 42) | 41 (19, 64) | 34 (17, 51) |
| KLgol, µM | 45±6 | 66±18 | 24±5 |
| Substrate Binding Stoichiometry, 3 mol/mol subunit |
|||
| ATP | 0.4±0.3 | 0.5±0.1 | nd |
| glycerol | 1.2±0.1 | 1.0±0.1 | nd |
| FBP inhibition | |||
| K0.5, µM | 630 (540, 720) | 470 (440, 500) | Not inhibited |
| W | 0.02 (0, 0.07) | 0.06 (0.03, 0.08) | |
| nH | 1.6 (1.3, 1.9) | 1.3 (1.2, 1.4) | |
| Sedimentation coefficients |
|||
| S20,w | 11.3±0.1 | 10.5±0.1 | 9.0±0.1 |
| S20,w + FBP | 11.4±0.1 | 11.2±0.1 | 10.1±0.1 |
1
Parameters were estimated by fits to the respective equations as described in the text with uncertainties given as 95% confidence limits shown in parenthesis or following the ± symbol, except as noted.
2
Parameters for EGK from [33] and for A65T EGK from [42]. KLgol was obtained from protection from inactivation by NEM with uncertainties given as the standard deviations. nd, not determined.
3
Ligand binding stoichiometries were determined as described under Materials and Methods. The uncertainties are shown as standard deviations.