Table 2.
Kinetic Analyses of Wild-Type and/or Mutant Forms of Hex S and B with MUG and MUGS Substrates
| forms of Hex | MUG Km (mM) | MUG Vmax [nmol h−1 (μg of total protein)−1] | MUG Vmax [nmol h−1 (μg of Hex)−1]a | MUGS Km (mM)b | MUGS Vmax [nmol h−1 (μg of total protein)−1]b | MUGS Vmax [nmol h−1 (μg of Hex)−1]a,b |
|---|---|---|---|---|---|---|
| pure Hex S: 2(αNR424) | 1.5 ± 0.2c | 2000 ± 50 | 2000 ± 50 | 0.3 ± 0.1 | 940 ± 40 | 940 ± 40 |
| WT Hex S: 2(αNR424) | 2.0 ± 0.2 | 155 ± 5 | 2200 | 0.45 ± 0.04 | 65 ± 2 | 940 |
| Hex S: 2(αNK424) | 2.1 ± 0.2 | 71 ± 2 | 3400 | 1.1 ± 0.1 | 28 ± 1 | 1350 |
| Hex S: 2(αDR424) | 2.4 ± 0.2 | 37 ± 1 | 3400 | 2.9 ± 0.5 | 15 ± 1 | 1350 |
| Hex S: 2(αNQ424) | 0.65 ± 0.05 | 110 ± 1 | 13000 | 4.1 ± 0.7 | 5.3 ± 0.6 | 620 |
| pure Hex B: 2(βDL454) | 0.71 ± 0.1 | 14500 ± 500 | 14500 ± 500 | NDd | ND | ND |
| at pH 3.0e | N/Af | N/A | N/A | 4.6 ± 0.6 | 710 ± 60 | 710 ± 60 |
a
Vmax values were adjusted to reflect the amount of Hex protein used in each assay (based on data from column 5, Table 1).
b
Assay was carried out at pH 4.2 for Hex S and at pH 3.0 for Hex B.
c
± are standard errors calculated from the curve-fitting program and do not include the experimental errors from determining either the initial protein or substrate concentrations which are estimated at an additional ~±10%.
d
Not determined. Assays produced only a linear response up to the limit after which substrate inhibition was observed at pH 3 (>3.5 mM; data not shown).
e
Assays were done at pH 3.0.
f
Not applicable.