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Related Experiment Videos

Methods for Determining Glycosyltransferase Kinetics.

Maria Ngo1, Michael D L Suits2

  • 1Department of Chemistry & Biochemistry, Wilfrid Laurier University, 75 University Avenue West, Waterloo, ON, Canada, N2L 3C5.

Methods in Molecular Biology (Clifton, N.J.)
|April 19, 2017
PubMed
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This study details colorimetric assays for quantifying glycosyltransferase activity, crucial for understanding bacterial cell wall synthesis and penicillin-binding proteins (PBPs). These methods enable precise kinetic characterization of these essential biosynthetic enzymes.

Area of Science:

  • Biochemistry
  • Enzymology
  • Microbiology

Background:

  • Glycosyltransferases are key biosynthetic enzymes catalyzing the transfer of activated monosaccharides.
  • Accurate kinetic characterization of glycosyltransferases is vital for understanding metabolic pathways.
  • Penicillin-binding proteins (PBPs) are essential targets in antibacterial drug development.

Purpose of the Study:

  • To provide an overview of colorimetric and coupled assay methods for glycosyltransferase activity determination.
  • To describe a specific protocol for quantifying glycosyltransferase activity in the context of penicillin-binding proteins (PBPs).
  • To enable indirect measurement of nucleotide-activated carbohydrate unit transfer.

Main Methods:

  • Utilized colorimetric assays detecting released products like para-nitrophenol.
Keywords:
Colorimetric assayEnzyme kineticsGlycosyltransferasesLipid II transferMalachite green dyepara-Nitrophenol

Related Experiment Videos

  • Employed coupled assays for sensitive inorganic phosphate detection.
  • Applied these techniques to measure glycosyltransferase activity in bacterial cell wall synthesis, specifically PBP-mediated lipid II transfer.

    • Main Results:

    • Established convenient and quantifiable kinetic characterization of glycosyltransferases.
    • Demonstrated the application of these assays for determining enzymatic activity through indirect product measurement.
    • Successfully applied the protocol to characterize PBP activity in bacterial murein chain elongation.

    Conclusions:

    • Colorimetric and coupled assays offer robust methods for glycosyltransferase kinetic characterization.
    • The described protocol is effective for assessing glycosyltransferase activity involved in bacterial cell wall synthesis.
    • This work facilitates further research into PBPs and the development of novel antibacterial strategies.