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Related Experiment Videos

Engineering the active site of ascorbate peroxidase.

A Celik1, P M Cullis, M J Sutcliffe

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

European Journal of Biochemistry
|December 20, 2000
PubMed
Summary

Recombinant pea cytosolic ascorbate peroxidase (rAPX) and its W41A variant were studied for thioether oxidation. The W41A variant showed significantly higher oxidation rates and enhanced enantioselectivity in sulfoxide production.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • Ascorbate peroxidases (APXs) are key enzymes in plant antioxidant systems.
  • Understanding their substrate specificity and catalytic mechanisms is crucial for biotechnological applications.
  • Site-directed mutagenesis offers a powerful tool to probe enzyme function and engineer novel properties.

Purpose of the Study:

  • To investigate the substrate oxidation of various thioethers by recombinant pea cytosolic ascorbate peroxidase (rAPX).
  • To characterize the enzymatic activity and enantioselectivity of a site-directed rAPX variant (W41A) with a modified active site.
  • To elucidate the structural basis for altered substrate specificity and stereoselectivity using computational modeling.

Main Methods:

  • Enzyme kinetics assays (kcat determination) for thioether oxidation by rAPX and W41A.

Related Experiment Videos

  • Analysis of enantiomeric excess of produced sulfoxides using chiral chromatography.
  • Isotopic labeling studies with H2(18)O2 to confirm oxygen transfer.
  • Structure-based computational modeling to rationalize experimental observations.

    • Main Results:

    • The W41A variant exhibited significantly higher catalytic rates (kcat) for thioether oxidation compared to wild-type rAPX (10-100 fold increase).
    • W41A demonstrated substantial enhancements in enantioselectivity, producing specific R:S ratios for various thioethers, unlike the racemic mixtures from rAPX.
    • Oxygen transfer from H2(18)O2 to thioethers was highly efficient (>95%) for both enzymes.
    • Computational modeling successfully rationalized the experimentally observed enantioselectivity, highlighting the role of Arg38 reorientation.

    Conclusions:

    • The distal W41 residue plays a critical role in modulating the substrate access and stereochemical outcome of thioether oxidation by rAPX.
    • Engineering of the rAPX active site, specifically the W41A mutation, can lead to improved catalytic efficiency and enantioselectivity.
    • Structural insights from modeling provide a quantitative understanding of how active site modifications influence enzyme function and stereoselectivity.