Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation

• Published in: Biochemical journal Vol. 328 ( Pt 3); no. 3; pp. 945 - 949
• Main Authors: Drouillard, S, Armand, S, Davies, G J, Vorgias, C E, Henrissat, B
• Format: Journal Article
• Language: English
• Published: England Portland Press 15.12.1997
• Subjects: Acetylation; Acetylglucosamine - analogs & derivatives; Acetylglucosamine - chemistry; Acetylglucosamine - metabolism; Acetylglucosaminidase - antagonists & inhibitors; Acetylglucosaminidase - metabolism; Carbohydrate Sequence; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Escherichia coli - genetics; Glycoside Hydrolases - metabolism; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Sequence Data; Oligosaccharides - chemistry; Oligosaccharides - pharmacology; Recombinant Proteins - metabolism; Serratia marcescens - enzymology; Stereoisomerism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3280945

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.