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 beta-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.
Klarskov
K
K
Department of Biochemistry, Physiology and Microbiology, University of Gent, Belgium.
Piens
K
K
Ståhlberg
J
J
Høj
P B
PB
Beeumen
J V
JV
Claeyssens
M
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004852:Epoxy Compounds / Q000494:pharmacology; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005960:Glucosides / Q000494:pharmacology; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D014242:Trichoderma / Q000201:enzymology*
D004791:Enzyme Inhibitors; D004852:Epoxy Compounds; D005656:Fungal Proteins; D005960:Glucosides; C081533:3,4-epoxybutyl-beta-cellobioside; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase