Concurrent enzyme reactions and binding events for chitinases interacting with chitosan oligosaccharides monitored by high resolution mass spectrometry

Abstract

High resolution nanoelectrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry was used to monitor formation of non-covalent complexes between chitinase B, a family 18 glycoside hydrolase, and hetero-chitooligosaccharides. Besides anticipated productive binding followed by glycoside hydrolysis, additional processes like transglycosylation, non-productive binding of potential inhibitors, and oxidation were detected by analysis of multiple non-covalent enzyme–ligand complexes as well as free oligosaccharide ions. Upon mutation of Asp142, responsible for binding and activation of the N-acetylgroup of the -1 sugar for nucleophilic attack on the anomeric carbon, a significant decrease of catalytic activity in the mutant protein was accompanied by drastic changes in oligosaccharide binding preferences and by changed reaction product profiles. The analysis of complex “mixed” enzyme–ligand interactions with unprecedented accuracy and level of detail also provided direct evidence for the occurrence of transglycosylation, leading to the formation of longer oligosaccharides in the reactions with both wild-type ChiB and its D142N mutant. This direct monitoring strategy of distinct types of enzyme–ligand interactions to identify in parallel all products of main and side reactions represents a general approach made possible by MS of ultrahigh resolution and mass accuracy.