The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B.
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 A 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.
Vuong
Thu V
TV
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.
Wilson
David B
DB
D000192:Actinomycetales / Q000201:enzymology*; D000596:Amino Acids / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary
D000596:Amino Acids; D001426:Bacterial Proteins; D002480:Cellulase; C506385:endoglucanase Cel6A, Thermobifida fusca