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

  • Darryl R. Jones
  • Richard McLean
  • D. Wade Abbott
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1588)

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.

Key words

Enzyme assay Kinetics Polysaccharide lyase (PL) Pectin Pectate Metal-dependent Uronic acid 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Darryl R. Jones
    • 1
  • Richard McLean
    • 1
    • 2
  • D. Wade Abbott
    • 1
    • 2
  1. 1.Functional Genomics of Complex Carbohydrate Utilization, Lethbridge Research and Development CentreAgriculture and Agri-Food CanadaLethbridgeCanada
  2. 2.Department of Chemistry and BiochemistryUniversity of LethbridgeLethbridgeCanada

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