Identification of the Catalytic Nucleophile of Endoglucanase I from Fusarium oxysporum by Mass Spectrometry
Authors:
- 1 May 1997
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 36 (19) , 5893-5901
Abstract
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-β-d-cellobioside results in the time-dependent inactivation of the enzyme (ki = 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.Keywords
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