Activating Compound | Comment | Organism | Structure |
---|---|---|---|
fructose 1,6-bisphosphate | mutant enzyme T298C shows no catalytic activity in the absence of the heterotrophic activator | Saccharomyces cerevisiae |
Protein Variants | Comment | Organism |
---|---|---|
T298C | no catalytic activity in the absence of the heterotrophic activator fructose 1,6-bisphosphate. In the presence of Mg2+ and D-fructose 1,6-bisphosphate, T298C has approximately 20% of the activity of wild-type enzyme. The activator constant for FBP increases by 1 order of magnitude compared to this constant with the wild type enzyme. T298C shows positive cooperativity by D-fructose 1,6-bisphosphate with a Hill coefficient of 2.6. Mn2+-activated T298C behaves like Mn2+-activated wild type enzyme with a Vmax that is 20% of that for the wild type enzyme with or without fructose-1,6-bisphosphate. A pH-rate profile of T298C relative to that for wild type enzyme shows that pKa2 has shifted from 6.4 in wild type to 5.5, indicating that the thiol group elicits an acidic pK shift. Inactivation of both wild type and T298C by iodoacetate elicits a pseudo-first-order loss of activity with T298C being inactivated from 8 to 100 times faster than wild-type enzyme. A pH dependence of the inactivation rate constant for T298C gives a value of pH 8.2, consistent with the pK for a thiol. Changes in fluorescence indicate that the T298C-Mg2+ complex binds PEP, ADP, and both ligands together. This demonstrates that the lack of activity is not due to the loss of substrate binding but to the lack of ability to induce the proper conformational change. The mutation also induces changes in binding of fructose-1,6-bisphosphate to all the relevant complexes. Binding of the metal and binding of phosphoenolpyruvate to the enzyme complexes are also differentially altered. Solvent isotope effects are observed for both wild type and T298C. Proton inventory studies indicate that kcat is affected by a proton from water in the transition state and the effects are metal ion-dependent. The results are consistent with water being the active site proton donor. Active site residue T298 is not critical for activity but plays a role in the activation of the water and affects the pK that modulates catalytic activity | Saccharomyces cerevisiae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.021 | - |
phosphoenolpyruvate | pH 6.2, wild-type enzyme, Mn2+- and fructose 1,6-bisphosphate activated | Saccharomyces cerevisiae | |
0.045 | - |
phosphoenolpyruvate | pH 6.2, wild-type enzyme, Mn2+-activated | Saccharomyces cerevisiae | |
0.066 | - |
phosphoenolpyruvate | pH 6.2, mutant enzyme T298C, Mn2+- and fructose 1,6-bisphosphate activated | Saccharomyces cerevisiae | |
0.242 | - |
phosphoenolpyruvate | pH 6.2, mutant enzyme T298C, Mn2+-activated | Saccharomyces cerevisiae | |
0.31 | - |
phosphoenolpyruvate | pH 6.2, wild-type enzyme, Mg2+ and fructose 1,6-bisphosphate activated | Saccharomyces cerevisiae | |
1.18 | - |
phosphoenolpyruvate | pH 6.2, wild-type enzyme, Mg2+-activated | Saccharomyces cerevisiae | |
4.43 | - |
phosphoenolpyruvate | pH 6.2, mutant enzyme T298C, Mg2+- and fructose 1,6-bisphosphate activated | Saccharomyces cerevisiae |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mn2+ | Mn2+-activated T298C behaves like Mn2+-activated wild type enzyme with a Vmax that is 20% of that for the wild type enzyme with or without D-fructose-1,6-bisphosphate | Saccharomyces cerevisiae |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | - |
- |
- |
Purification (Comment) | Organism |
---|---|
mutant enzyme T298C | Saccharomyces cerevisiae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ADP + phosphoenolpyruvate | - |
Saccharomyces cerevisiae | ATP + pyruvate | - |
? |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
12.1 | - |
phosphoenolpyruvate | pH 6.2, mutant enzyme T298C, Mn2+-activated | Saccharomyces cerevisiae | |
13.9 | - |
phosphoenolpyruvate | pH 6.2, mutant enzyme T298C, Mn2+- and fructose 1,6-bisphosphate activated | Saccharomyces cerevisiae | |
41.3 | - |
phosphoenolpyruvate | pH 6.2, mutant enzyme T298C, Mg2+- and fructose 1,6-bisphosphate activated | Saccharomyces cerevisiae | |
58.4 | - |
phosphoenolpyruvate | pH 6.2, wild-type enzyme, Mn2+-activated | Saccharomyces cerevisiae | |
66 | - |
phosphoenolpyruvate | pH 6.2, wild-type enzyme, Mn2+- and fructose 1,6-bisphosphate activated | Saccharomyces cerevisiae | |
226 | - |
phosphoenolpyruvate | pH 6.2, wild-type enzyme, Mg2+ and fructose 1,6-bisphosphate activated | Saccharomyces cerevisiae | |
232 | - |
phosphoenolpyruvate | pH 6.2, wild-type enzyme, Mg2+-activated | Saccharomyces cerevisiae |