Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates.
Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.
Rye
Carl S
CS
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
Withers
Stephen G
SG
D002384:Catalysis; D002808:Chondroitin Lyases / Q000737:chemistry / Q000378:metabolism*; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005459:Fluorides / Q000032:analysis; D017736:Ion-Selective Electrodes; D007554:Isotopes; D007700:Kinetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013816:Thermodynamics
D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D007554:Isotopes; D002808:Chondroitin Lyases; D005459:Fluorides