Stereochemistry of the hydrolysis reaction catalyzed by endoglucanase Z from Erwinia chrysanthemi.
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.
Barras
F
F
Laboratoire de Chimie Bactérienne, CNRS, Marseille, France.
Bortoli-German
I
I
Bauzan
M
M
Rouvier
J
J
Gey
C
C
Heyraud
A
A
Henrissat
B
B
D002384:Catalysis; D002480:Cellulase / Q000737:chemistry* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D016974:Dickeya chrysanthemi / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D009844:Oligosaccharides / Q000378:metabolism; D011487:Protein Conformation; D012995:Solubility; D013379:Substrate Specificity; D013402:Sugar Alcohols / Q000378:metabolism
D009844:Oligosaccharides; D013402:Sugar Alcohols; C074087:cellotetraitol; D002480:Cellulase