Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi

FEBS Lett. 1992 Mar 30;300(2):145-8. doi: 10.1016/0014-5793(92)80183-h.

Abstract

Endoglucanase Z from the phytopathogenic bacterium Erwinia chrysanthemi (strain 3937) was purified by affinity chromatography on microcrystalline cellulose Avicel PH101. A kinetic characterization using p-nitrophenyl beta-D-cellobioside and p-nitrophenyl beta-D-lactosde as substrates was conducted: endoglucanase Z exhibited Km values of 3 mM and 7.5 mM and Vm values of 129 and 40 nmol.min-1.mg-1 towards p-nitrophenyl beta-D-cellobioside (kcat = 0.1 s-1) and p-nitrophenyl beta-D-lactoside (kcat = 0.03 s-1), respectively). The hydrolysis of cellotetraitol by endoglucanase Z was followed by HPLC and 1H NMR. Results show that cellobiitol and beta-cellobiose are initially formed, demonstrating that the enzyme is acting by a molecular mechanism retaining the anomeric configuration. This suggests the involvement of a glycosyl-enzyme intermediate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalysis
  • Cellulase / chemistry*
  • Cellulase / isolation & purification
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Dickeya chrysanthemi / enzymology*
  • Escherichia coli / genetics
  • Hydrolysis
  • Magnetic Resonance Spectroscopy
  • Oligosaccharides / metabolism
  • Protein Conformation
  • Solubility
  • Substrate Specificity
  • Sugar Alcohols / metabolism

Substances

  • Oligosaccharides
  • Sugar Alcohols
  • cellotetraitol
  • Cellulase