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Phosphonate analogue substrates for enolase.

V E Anderson1, W W Cleland

  • 1Institute for Enzyme Research, University of Wisconsin, Madison 53705.

Biochemistry
|November 20, 1990
PubMed
Summary

Yeast enolase exhibits slow kinetics with phosphonate analogues due to a more stable carbanion intermediate. This increased stability, caused by replacing oxygen with carbon, affects both substrate binding and product formation rates.

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Organic chemistry

Background:

  • Yeast enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate.
  • Phosphonate analogues are often used to probe enzyme mechanisms.
  • The role of metal ions and protonation states in enolase catalysis is crucial.

Purpose of the Study:

  • To investigate the kinetic behavior of yeast enolase with phosphonate analogues.
  • To elucidate the mechanism of substrate binding and catalysis involving these analogues.
  • To understand the factors contributing to the observed slow reaction rates.

Main Methods:

  • Enzyme kinetic assays were performed on yeast enolase using a methylene analogue of 2-phosphoglycerate.
  • pH-dependent kinetic parameters were measured to determine substrate and metal ion binding requirements.
  • Primary deuterium isotope effects and D2O solvent isotope effects were utilized to probe rate-limiting steps.

Main Results:

  • The phosphonate analogue binds to yeast enolase as a dianion, requiring unprotonated catalytic sites for subsequent Mg2+ binding.
  • Proton removal is rate-limiting for substrate binding (V/KMg), while carbanion breakdown is rate-limiting for catalysis (V).
  • A significant D2O solvent isotope effect on the reverse reaction suggests increased stability of the carbanion intermediate.

Conclusions:

  • The slow reaction rates observed with phosphonate analogues are attributed to a more stable carbanion intermediate compared to natural substrates.
  • The chemical modification (replacing oxygen with carbon at C-2) leads to this enhanced carbanion stability, impacting catalytic efficiency.
  • These findings provide insights into the enolase catalytic mechanism and the role of intermediate stability.

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