TABLE 2.
Catalytic and allosteric properties of GlpK mutants
| GlpK residue change | Catalytic parametersa |
FBP inhibitionb |
IIAGlc inhibitionb |
Molecular weightc |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Vmax | Km ATP | Vmax/Km | SAo | W | nH | K0.5 | SAo, | W | K0.5 | Mr, app | |
| unit/mg | μm | L* min−1mg−1 | unit/mg | mm | unit/mg | μm | |||||
| V61L | 18 ± 0.4 | 6 ± 0.6 | 3 ± 0.1 | 55 ± 1 | NA | NA | >100 | 56 ± 1 | 0.12 ± 0.04 | 13 ± 2 | 114,000 |
| D72V | 24 ± 0.4 | 9 ± 1 | 2.7 ± 0.1 | 53 ± 1 | 0.015 | 1.3 | 21 ± 2 | 53 ± 1 | 0.07 ± 0.01 | 10 ± 1 | 138,000 |
| M271I | 77 ± 3 | 250 ± 20 | 0.31 ± 0.1 | 54 ± 3 | 0.015 | 1.3 | 16 ± 1 | 69 ± 1 | 0.23 ± 0.03 | 37 ± 4 | NDc |
| Q37P | 86 ± 3 | 190 ± 20 | 0.45 ± 0.1 | 80 ± 1 | 0.015 | 1.3 | 3.2 ± 0.1 | 80 ± 1 | 0.08 ± 0.03 | 10 ± 1 | 152,000 |
| N-termd | 16.2 ± 0.4 | 8.5 ± 0.8 | 1.9 ± 0.1 | 62 ± 1 | 0.015 ± 0.02 | 1.3 ± 0.1 | 0.52 ± 0.01 | 62 ± 1 | 0.01 ± 0.01 | 6 ± 0.6 | 162,000 |
| Native GlpK | 18.1 ± 0.4 | 7.8 ± 0.7 | 2.3 ± 0.1 | 62 ± 2 | 0.03 ± 0.026 | 1.7 ± 0.2 | 0.52 ± 0.04 | 64 ± 1 | 0.09 ± 0.02 | 6 ± 0.6 | 158,000 |
| Spearman re | 0.0 | −0.3 | 0.6 | −0.6 | 1.00 | −0.6 | 0.5 | 0.6 | −1.00 | ||
| p value | 0.0167 | 0.0833 | |||||||||
a Catalytic parameters are obtained from fits of the dependence of the initial velocity on [ATP] in the saturating presence of glycerol (10 mm) to the Michaelis-Menten equation. All values on chart are shown ± S.E.
b FBP and IIAGlc inhibition are determined at 2.5 mm ATP and 10 mm glycerol. SAo is the specific activity at 0 allosteric inhibitor and W is given by the ratio SA∞/SAo, where SA∞ is the specific activity in the saturating presence of the inhibitor. FBP inhibition shows cooperative homotropic effects, given by the Hill coefficient, nH. K0.5 is the concentration of inhibitor that gives one-half of the maximum inhibition. For fits of FBP inhibition for the variant enzymes, the values for W and nH were fixed to the values obtained for the enzyme with the N-terminal extension.
c Molecular weight of oligomeric GlpK as it exists in solution was measured by gel-permeation chromatography. The apparent molecular mass of the M271I mutant could not be determined because of its non-specific interaction with the column matrix. A similar problem was encountered for the G304S mutant, which disrupts the same residue interaction (25).
d Native GlpK enzyme carrying the same N-terminal extension (Gly-Pro-Leu-Gly-Ser-Pro-Glu-Phe) as the GlpK mutant enzymes, which remains after cleavage of the GST tag used to purify the proteins (See “Experimental Procedures” and supplemental notes).
e Correlation between the measured parameters and relative fitness imparted by the mutations is estimated using the non-parametric Spearman correlation coefficient, which was applied to parameters with measured differences across the mutants. The p-value is shown if the absolute value of Spearman r > 0.9. Measurements of the N-terminal wild type GlpK protein were used to calculate the coefficients.