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An Improved Kinetic Assay for the Characterization of Metal-Dependent Pectate Lyases

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Protein-Carbohydrate Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1588))

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Abstract

Pectate lyases are a subset of polysaccharide lyases (PLs) that specifically utilize a metal dependent β-elimination mechanism to cleave glyosidic bonds in homogalacturonan (HG; α-d-1,4-galacturonic acid). Most commonly, PLs harness calcium for catalysis; however, some PL families (e.g., PL2 and PL22) display preferences for transitional metals. Deploying alternative metals during β-elimination is correlated with signature coordination pocket chemistry, and is reflective of the evolution, functional specialization, and cellular location of PL activity. Here we describe an optimized method for the analysis of metal-dependent polysaccharide lyases (PLs). We use an endolytic PL2 from Yersinia enterocolitica (YePL2A) as example to demonstrate how altering the catalytic metal within the reaction can modulate PL kinetics.

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Author information

Authors and Affiliations

  1. Functional Genomics of Complex Carbohydrate Utilization, Lethbridge Research and Development Centre, Agriculture and Agri-Food Canada, 5403 1st Ave. South, Lethbridge, T1J 4B1, AB, Canada

    Darryl R. Jones, Richard McLean & D. Wade Abbott

  2. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, T1K 3M4, AB, Canada

    Richard McLean & D. Wade Abbott

Authors
  1. Darryl R. Jones
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  2. Richard McLean
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  3. D. Wade Abbott
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Corresponding author

Correspondence to D. Wade Abbott .

Editor information

Editors and Affiliations

  1. Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada

    D. Wade Abbott

  2. Biotechnology Institute, University of Groningen, Groningen, The Netherlands

    Alicia Lammerts van Bueren

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Jones, D.R., McLean, R., Abbott, D.W. (2017). An Improved Kinetic Assay for the Characterization of Metal-Dependent Pectate Lyases. In: Abbott, D., Lammerts van Bueren, A. (eds) Protein-Carbohydrate Interactions. Methods in Molecular Biology, vol 1588. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6899-2_4

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  • DOI: https://doi.org/10.1007/978-1-4939-6899-2_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6898-5

  • Online ISBN: 978-1-4939-6899-2

  • eBook Packages: Springer Protocols

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