The pK(sub a) of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of Bacillus circulans xylanase

Selective isotopic labelling and 13C-NMR studies on the pK(sub a) values of the catalytic glutamic acid residues in wild type/mutant Bacillus circulans (BCX) and subtilis xylanases reveal a two-step hydrolysis mechanism involving a covalent glycosyl-enzyme intermediate. The double-displacement reaction of BCX may be broken into glycosylation and deglycosylation steps. The glycosylation step produces the glycosyl-enzyme intermediate and deglycosylation regenerates the native enzyme. These protonation and deprotonation steps are governed by glutamic ionization states.