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 wal l beta-glucans via a double-displacement mechanism involving a glycosy l enzyme intermediate. Reaction of the enzyme with itrophenyl-2-deoxy- 2-fluoro-beta-n-glucopyranoside resulted in the time-dependent inactiv ation of this enzyme via the accumulation of a 2-deoxy-2-fluoro-glycos yl-enzyme intermediate as monitored also by electrospray mass spectrom etry, The catalytic competence of this intermediate is demonstrated by its reactivation through hydrolysis (k(react) = 0.0019 min(-1)) and b y transglycosylation to benzyl thio-beta-D-glucopyranoside (k(react) = 0.024 min(-1); K-react 56 mM). Peptic digestion of the labeled enzyme followed by tandem mass spectrometric analysis in the neutral loss mo de allowed detection of two glycosylated active site peptides, the seq uences of which were identified as NVAGEW and NVAGEWSAA. A crucial rol e for Glu-330 is confirmed by site directed mutagenesis at this site a nd kinetic analysis of the resultant mutant, The activity of the Glu-3 30 --> Gln mutant is reduced over 50,000-fold compared to the wild typ e enzyme. The glutamic acid, identified in the exoglucanase as Glu-330 , is completely conserved in this family of enzymes and is hereby iden tified as the catalytic nucleophile.