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Related Experiment Videos

Interactions between citrate and nucleoside triphosphates in binding to phosphofructokinase.

Y Ogawa, D E Atkinson

    Biochemistry
    |February 12, 1985
    PubMed
    Summary

    Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) enhance citrate binding to phosphofructokinase, influencing enzyme activity. GTP binding at the catalytic site increases citrate affinity without inhibiting enzyme function.

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    Area of Science:

    • Biochemistry
    • Enzymology
    • Protein-ligand interactions

    Background:

    • Phosphofructokinase (PFK) is a key regulatory enzyme in glycolysis.
    • Understanding the allosteric regulation of PFK by nucleotides and metabolites is crucial for metabolic control.
    • Investigating the binding kinetics of various ligands provides insight into enzyme mechanisms.

    Purpose of the Study:

    • To investigate the interrelationships between the binding of citrate, ATP, GTP, and AMP-PNP to rabbit muscle phosphofructokinase.
    • To elucidate the role of GTP binding at the catalytic site in modulating citrate binding.
    • To determine the binding sites and affinities of ATP and GTP on the enzyme.

    Main Methods:

    • Enzyme binding assays were performed using rabbit muscle phosphofructokinase.

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  • Pyruvate kinase and phosphoenolpyruvate were used to maintain ADP levels at 25°C.
  • Citrate binding was measured in the presence of varying concentrations of ATP, GTP, and AMP-PNP.

    • Main Results:

    • GTP enhanced citrate binding to phosphofructokinase similarly to ATP, despite GTP not inhibiting catalytic activity.
    • Adenosine triphosphate (ATP) and guanosine triphosphate (GTP) appear to bind at three sites per enzyme subunit, with the third site exhibiting weaker binding.
    • Dissociation constants indicated that GTP does not effectively compete for the inhibitory site, aligning with kinetic data.

    Conclusions:

    • GTP binding at the catalytic site of phosphofructokinase enhances citrate binding, suggesting a regulatory role beyond simple catalytic inhibition.
    • The binding of ATP, GTP, and AMP-PNP influences citrate affinity, highlighting complex allosteric regulation of phosphofructokinase.
    • While multiple binding sites exist, precise affinity estimations are challenging due to the nature of multi-site binding kinetics.