Journal
FEBS JOURNAL
Volume 276, Issue 14, Pages 4261-4269Publisher
WILEY
DOI: 10.1111/j.1742-4658.2009.07097.x
Keywords
catalytic base; exocellulase; glycoside hydrolase; hydrolysis mechanism; proton-transferring network
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Funding
- Vietnam Education Foundation
- US Department of Energy [DE-FG-022179-16-18]
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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 angstrom 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.
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