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Related Experiment Videos

Chorismate lyase: kinetics and engineering for stability.

M J Holden1, M P Mayhew, D T Gallagher

  • 1National Institute of Standards and Technology, Chemical Science and Technology Laboratory, Biotechnology Division, 100 Bureau Drive, Gaithersburg, MD 20899-8312, USA. marcia.holden@nist.gov

Biochimica Et Biophysica Acta
|February 5, 2002
PubMed
Summary

Chorismate lyase (CL) produces p-hydroxybenzoate for ubiquinone synthesis. Modifying surface cysteines improves CL stability and enzyme behavior, crucial for understanding ubiquinone biosynthesis.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Chorismate lyase (CL) catalyzes a key step in ubiquinone biosynthesis by converting chorismate to p-hydroxybenzoate (p-HB).
  • Wild-type CL exhibits significant protein aggregation, hindering its study and potential applications.

Purpose of the Study:

  • To investigate the structural and kinetic properties of chorismate lyase (CL).
  • To identify and address the causes of protein aggregation in CL.
  • To elucidate the mechanism of product inhibition and active site interactions.

Main Methods:

  • Spectroscopic and chemical analyses.
  • Kinetic measurements (kcat, Km, Kp) using initial rate and progress curve methods.
  • Site-directed mutagenesis and chemical modification of cysteine residues.

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  • Inhibition studies with substrate and product analogs.

    • Main Results:

    • CL kinetics were characterized (kcat=1.7 s⁻¹, Km=29 μM) with significant product inhibition by p-HB (Kp=2.1 μM).
    • Protein aggregation was primarily attributed to two surface-active cysteines; their modification or mutation improved solution behavior with minimal impact on activity.
    • Progress curve methods proved more efficient for analyzing enzyme kinetics due to stable enzyme-product complex formation.

    Conclusions:

    • Surface cysteine residues are responsible for CL aggregation and can be modified to improve enzyme stability.
    • Product inhibition by p-HB is a significant feature of CL activity.
    • The biological role of product retention may involve directed delivery to downstream enzymes in the ubiquinone pathway.