Glutamic acid 274 is the nucleophile in the active site of a "retaining" exoglucanase from Cellulomonas fimi.
In addition to its known substrate activity with p-nitrophenyl beta-cellobioside, the exoglucanase from Cellulomonas fimi has been shown to utilize substituted phenyl beta-glucosides as substrates, of which the best is 2',4'-dinitrophenyl beta-D-glucopyranoside. The enzyme can be inactivated by treatment with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-glucopyranoside, by trapping of the covalent intermediate in catalysis, as has been shown for a beta-glucosidase (Withers, S.G., and Street, I.P. (1988) J. Am. Chem. Soc. 110, 8551-8553). The intermediate formed is stable but can undergo turnover in the presence of cellobiose, reactivating the enzyme by transglycosylation. Using a tritium-labeled inactivator it has been possible to isolate and sequence a radiolabeled peptide from this enzyme, and the active site nucleophile has been identified as glutamic acid residue 274. This glutamic acid residue and its sequentially proximal amino acids are absolutely conserved in the homologous family F of cellulases.
Tull
D
D
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Withers
S G
SG
Gilkes
N R
NR
Kilburn
D G
DG
Warren
R A
RA
Aebersold
R
R
D000192:Actinomycetales / Q000201:enzymology*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002480:Cellulase / Q000235:genetics; D004789:Enzyme Activation; D043326:Glucan 1,3-beta-Glucosidase; D005960:Glucosides / Q000737:chemistry; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D006026:Glycoside Hydrolases / Q000235:genetics; D008969:Molecular Sequence Data; D043325:Xylan Endo-1,3-beta-Xylosidase; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D005960:Glucosides; D005971:Glutamates; C070512:2',4'-dinitrophenylglucopyranoside; D018698:Glutamic Acid; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; D043325:Xylan Endo-1,3-beta-Xylosidase; D002480:Cellulase; D043326:Glucan 1,3-beta-Glucosidase