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-lactoside as substrates was conducted: endoglucanase Z exhibited K(m) values of 3 mM and 7.5 mM and V(m) values of 129 and 40 nmol.min-1.mg-1 towards p-nitrophenyl beta-D-cellobioside (k(cat) = 0.1 s-1) and p-nitrophenyl beta-D-lactoside (k(cat) = 0.03 s-1), respectively). The hydrolysis of cellotetraitol by endoglucanase Z was followed by HPLC and H-1 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.