|Title||ENZYMATIC CLEAVAGE OF GLYCOSIDES - HOW DOES IT HAPPEN|
|Publication Type||Journal Article|
|Year of Publication||1995|
|Journal||PURE AND APPLIED CHEMISTRY|
Many glycosidases operate through a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolysed via oxocarbenium ion-like transition states with acid/base catalysis. The two key active site residues involved in this mechanism, the active site nucleophile and the acid/base catalyst have been identified by novel means. The nucleophile is identified using a mechanism-based inactivator which functions by formation of a stabilised glycosyl-enzyme intermediate. Identification of the labelled peptide from proteolytic hydrolysates is achieved using a new tandem mass spectrometric method. The acid/base catalyst is identified by detailed kinetic analysis of candidate amino acids chosen on the basis of sequence similarities.