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Literature summary for 2.7.1.40 extracted from

  • Susan-Resiga, D.; Nowak, T.
    Proton donor in yeast pyruvate kinase: chemical and kinetic properties of the active site Thr 298 to Cys mutant (2004), Biochemistry, 43, 15230-15245.
    View publication on PubMed
Search for more literature for 2.7.1.40

Activating Compound

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

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 [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

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

Organism UniProt Comment Textmining
Saccharomyces cerevisiae
-
-
-

Purification (Commentary)

Purification (Comment) Organism
mutant enzyme T298C Saccharomyces cerevisiae

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ADP + phosphoenolpyruvate
-
 Saccharomyces cerevisiae ATP + pyruvate
-
 ?

Turnover Number [1/s]

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