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The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B
Author(s) -
Vuong Thu V.,
Wilson David B.
Publication year - 2009
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2009.07097.x
Subject(s) - hydrolysis , catalysis , residue (chemistry) , chemistry , glycoside hydrolase , stereochemistry , enzyme , biochemistry , mutagenesis , mutant , gene
Thermobifida fusca exocellulase Cel6B acts by an inverting hydrolysis mechanism; however, the catalytic acid and base residues for this enzyme have not been confirmed. Site‐directed mutagenesis and kinetic studies were used to show that Asp274 is the catalytic acid, which is consistent with what is found for other members of family‐6 glycoside hydrolases; however, a single catalytic base was not identified. Mutation of all putative catalytic base residues, within 6 Å of the −1/+1 glucose subsites, including the highly conserved Asp226, Asp497 and Glu495, as well as Ser232 and Tyr220, did not reveal a catalytic base, although these residues are all important for activity. We propose a novel hydrolysis mechanism for T. fusca Cel6B involving a proton‐transferring network to carry out the catalytic base function.