Cellobiohydrolase I from Trichoderma reesei: Identification of an active-site nucleophile and additional information on sequence including the glycosylation pattern of the core protein

(R,S)-3,4-Epoxybutyl β-cellobioside, but not the corresponding propyl and pentyl derivatives, inactivates specifically and irreversibly cellobiohydrolase I from Trichoderma reesei by covalent modification of Glu212, the putative active-site nucleophile. The position and identity of the modified amino acid residue were determined using a combination of comparative liquid chromatography coupled on-line to electrospray ionization mass spectrometry, tandem mass spectrometry and microsequencing. It was found that the core protein corresponds to the N-terminal sequence pyrGlu1-Gly434(Gly435) of intact cellobiohydrolase I. In the particular enzyme samples investigated, the asparagine residues in positions 45, 270 and 384 are each linked to a single 2-acetamido-2-deoxy-d-glucopyranose residue. (R,S)-3,4-Epoxybutyl β-cellobioside inactivates cellobiohydrolase I from T. reesei by covalent modification of Glu212, the putative active site nucleophile.