Identification of the catalytic nucleophile of endoglucanase I from Fusarium oxysporum by mass spectrometry.
The endoglucanase EG I from Fusarium oxysporum catalyzes the hydrolysis of cellulose via a double-displacement mechanism involving the formation and hydrolysis of a glycosyl-enzyme intermediate. Treatment of EG I with 2',4'-dinitrophenyl-2-deoxy-2-fluoro-beta-D-cellobioside results in the time-dependent inactivation of the enzyme (k(i) = 1.36 min(-1), Ki = 0.88 mM) via trapping of a covalent 2-deoxy-2-fluorocellobiosyl-enzyme intermediate. This intermediate is, however, catalytically competent undergoing transglycosylation, thus reactivation, in the presence of D-cellobiose. Analysis of a peptic digest of the inactivated enzyme by HPLC/ESMS/MS in the neutral loss mode allowed identification of a 2-fluorocellobiosyl-labeled peptide containing Glu197. This was confirmed by comparative mapping studies and subsequent Edman degradation analysis. This residue is completely conserved in glycosidase family 7, to which EG I belongs, consistent with its key role as the catalytic nucleophile.
Mackenzie
L F
LF
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Davies
G J
GJ
Schülein
M
M
Withers
S G
SG
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002480:Cellulase / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism
D005656:Fungal Proteins; D010455:Peptides; D018698:Glutamic Acid; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase