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-beta-D-cellobioside results in the time-dependent inactivation of the enzyme (k(i) = 1.36 min(-1), K-i = 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.
| Original language | English |
|---|---|
| Pages (from-to) | 5893-5901 |
| Number of pages | 9 |
| Journal | Biochemistry |
| Volume | 36 |
| Issue number | 19 |
| Publication status | Published - 13 May 1997 |
Keywords
- ACTIVE-SITE NUCLEOPHILE
- ACID-SEQUENCE SIMILARITIES
- BETA-GLUCOSIDASE
- CLOSTRIDIUM-THERMOCELLUM
- ENZYME INTERMEDIATE
- CRYSTAL-STRUCTURE
- MECHANISMS
- CELLULASES
- CLASSIFICATION
- GLYCOSIDASE