Table 3.
Substrate effects on dimerization constants for enzyme I and the active-site mutant (EI(H189A): sedimentation equilibrium studiesa
| Enzyme I (−/+ mM effectors) | Log K1,2′ (M monomer)−1 20°C |
| Dephospho-EI (wt) | 5.31 ± 0.20b |
| Hepes substituted for phosphate in buffer | 5.58 ± 0.09 (9) |
| (+5 pyruvate) | 5.12 ± 0.03b |
| (+0.65 phosphonopyruvate/2 Mg2+) | 6.93 ± 0.07 (6) |
| EI(H189A) | 5.49 ± 0.04b |
| Hepes substituted for phosphate in buffer | 5.76 ± 0.12 (9) |
| (+2 Mg2+) | 5.30 ± 0.08b |
| (+5 Pyr/2 Mg2+) | 4.16 ± 0.11 (9) |
| (+5 Pyr/0.20 PEP/2 Mg2+) | 5.67 ± 0.08 (8) |
| (+5 Pyr/1 PEP/2 Mg2+) | 7.4 ± 0.5 (6) |
| (+1 PEP/2 Mg2+) | ≥108 b,c |
a Buffer (−/+ effectors) used for equilibration of proteins prior to and during analytical ultracentrifugation was 20 mM potassium phosphate, 100 mM KCl, and 2 mM ME, pH 7.5, except in the two cases in which 20 mM Hepes/KOH was substituted for phosphate. Values of the dimerization constant expressed per M monomer (log K1,2′) are averaged from global analysis of 6–9 data files (as indicated in parentheses for new data), each having three concentrations of protein (−0.3, 0.5, 0.8 mg/mL), after 24–44 h at 10,000 rpm, 20°C (see Materials and Methods).
b From Dimitrova et al. (2002) and included for comparison with the new results.
c The value given for log K1,2′ ≥8 in Table 2 simply represents the limits in the sensitivity of absorbance measurements in sedimentation equilibrium scans at 280 nm.