Identification of Glu-330 as the catalytic nucleophile of Candida albicans exo-beta-(1,3)-glucanase.

The exo-beta-(1,3)-glucanase from Candida albicans hydrolyzes cell wall beta-glucans via a double-displacement mechanism involving a glycosyl enzyme intermediate. Reaction of the enzyme with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-glucopyranoside resulted in the time-dependent inactivation of this enzyme via the accumulation of a 2-deoxy-2-fluoro-glycosyl-enzyme intermediate as monitored also by electrospray mass spectrometry. The catalytic competence of this intermediate is demonstrated by its reactivation through hydrolysis (kreact = 0.0019 min-1) and by transglycosylation to benzyl thio-beta-D-glucopyranoside (kreact = 0.024 min-1; Kreact = 56 mM). Peptic digestion of the labeled enzyme followed by tandem mass spectrometric analysis in the neutral loss mode allowed detection of two glycosylated active site peptides, the sequences of which were identified as NVAGEW and NVAGEWSAA. A crucial role for Glu-330 is confirmed by site-directed mutagenesis at this site and kinetic analysis of the resultant mutant. The activity of the Glu-330 --> Gln mutant is reduced over 50,000-fold compared to the wild type enzyme. The glutamic acid, identified in the exoglucanase as Glu-330, is completely conserved in this family of enzymes and is hereby identified as the catalytic nucleophile.