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Related Experiment Videos

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.

Latesh Lad1, Martin Mewies, Emma Lloyd Raven

  • 1Department of Chemistry, University of Leicester, UK.

Biochemistry
|November 13, 2002
PubMed
Summary

Recombinant soybean cytosolic ascorbate peroxidase (rsAPX) exhibits two kinetically competent ascorbate binding sites, influencing its catalytic mechanism. Modifying Cys32 alters substrate binding, affecting reaction kinetics and eliminating sigmoidal behavior.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Plant Science

Background:

  • Ascorbate peroxidase (APX) is crucial for plant antioxidant defense.
  • Understanding the catalytic mechanism of APX is vital for elucidating plant stress responses.

Purpose of the Study:

  • To investigate the catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX).
  • To examine the role of Cys32 in ascorbate binding and catalytic activity.
  • To elucidate the kinetics of Compound I and Compound II reduction by L-ascorbate.

Main Methods:

  • Pre-steady-state and steady-state kinetic techniques were employed.
  • Spectroscopic analysis (wavelength maxima) was used to characterize intermediates.
  • Kinetic traces were analyzed at varying L-ascorbate concentrations.

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Main Results:

  • Compound I formation and reduction by substrate are rapid.
  • Reduction of Compound II by L-ascorbate is rate-limiting and shows biphasic kinetics above 500 microM.
  • Modification of Cys32 resulted in monophasic Compound II reduction kinetics and loss of sigmoidal substrate dependence.

Conclusions:

  • The data suggest two kinetically competent binding sites for ascorbate in rsAPX.
  • Cys32 plays a role in ascorbate binding and influences the overall catalytic mechanism.
  • The observed sigmoidal kinetics in wild-type rsAPX indicate cooperative binding or a regulatory mechanism involving ascorbate.