Abstract
The stereochemistry of the reaction catalysed by Serratia marcescens
chitobiase was determined by HPLC separation of the anomers of
N-acetylglucosamine produced during the hydrolysis of p-nitrophenyl
N-acetyl-beta-D-glucosaminide (PNP-GlcNAc). In the early stages of the
reaction, the beta-anomer was found to prevail, whereas the alpha-anomer
dominated at mutarotation equilibrium. This established that chitobiase
hydrolyses glycosidic bonds with overall retention of the anomeric
configuration. Chitobiase-catalysed hydrolysis of PNP-GlcNAc was
competitively inhibited by a series of chito-oligosaccharides (degree of
polymerization 2-5) that were selectively de-N-acetylated at their
non-reducing end. The results are in accord with the participation of
the acetamido group at C-2 of the substrate in the catalytic mechanism
of chitobiase and related enzymes.