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Proton transfer in catalysis by fumarase.

I A Rose1, J V Warms, D J Kuo

  • 1Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.

Biochemistry
|October 20, 1992
PubMed
Summary

Intermolecular transfer from malate to fumarate was observed. Slow dissociation of enzyme-malate complexes suggests a proton relay system at the active site, influencing reaction rates.

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Chemical reaction mechanisms

Background:

  • Enzyme catalysis involves complex intermediate steps.
  • Understanding substrate-enzyme interactions is crucial for elucidating reaction pathways.

Purpose of the Study:

  • To investigate the mechanism of intermolecular transfer between malate and fumarate.
  • To characterize the dissociation rates of enzyme-bound intermediates.
  • To explore the role of buffer in proton transfer during enzymatic reactions.

Main Methods:

  • Utilized 3T[14C]malate to trace intermolecular transfer.
  • Analyzed the effect of varying buffer concentrations and basicity on reaction rates.
  • Compared equilibrium exchange rates to determine substrate accessibility.

Main Results:

  • Demonstrated intermolecular transfer from malate to fumarate.
  • Observed slow dissociation of enzyme-triose (ET)-malate complexes, facilitating ET-malate formation.
  • Found that buffer concentration and basicity significantly influenced hydrogen dissociation rates.
  • Proton transfer to the medium was enhanced by buffer, suggesting a proton relay system.

Conclusions:

  • The enzyme-malate complex is functional, with its dissociation rate determining the V/Km of malate.
  • The abstracted hydroxyl group is readily exchanged with the medium, unlike the abstracted proton.
  • A proton relay system likely connects the active site to a remote site interfacing with water.

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