Catalytic and substrate-binding domains of endoglucanase 2 from Bacteroides succinogenes.
Endoglucanase 2 (EG2) of the cellulolytic ruminal anaerobe Bacteroides succinogenes is a 118-kilodalton (kDa) enzyme which binds to cellulose and produces cellotetraose as the end product of hydrolysis. The purified enzyme was treated with the protease trypsin in an attempt to isolate peptides which retained the ability to either hydrolyze soluble carboxymethyl cellulose or bind to insoluble cellulose. There was no loss in endoglucanase activity (carboxymethylcellulase) over a period of 2 h following the addition of trypsin. In comparison, there was a greater than eightfold reduction in the binding of carboxymethylcellulase activity to crystalline cellulose. A Lineweaver-Burk plot with amorphous cellulose as the substrate revealed that the trypsin-digested enzyme had an identical Vmax but a 1.9-fold-lower Km in comparison with the intact enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the trypsin-digested enzyme revealed two major peptides of 43 and 51 kDa (p43 and p51). The 43-kDa peptide was able to bind to both amorphous and crystalline cellulose, whereas p51 did not. Purified p51 had a molar activity toward carboxymethyl cellulose which was identical to that of the intact enzyme, but activity toward both amorphous and crystalline cellulose was reduced approximately twofold. Two high-titer monoclonal antibodies from mice immunized with the intact protein recognized p43 but not p51. The results are consistent with a bifunctional organization of EG2, in which the 118-kDa enzyme is composed of a 51-kDa catalytic domain and a highly antigenic 43-kDa substrate-binding domain. In terms of its domain structure and activity toward cellulose, EG2 is very similar to cellobiohydrolase II of Trichoderma reesei.
McGavin
M
M
Department of Microbiology, University of Guelph, Ontario, Canada.
Forsberg
C W
CW
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000648:ultrastructure; D001439:Bacteroides / Q000201:enzymology*; D001665:Binding Sites; D002480:Cellulase / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D000939:Epitopes; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D010446:Peptide Fragments; D002482:Cellulose; C056373:endoglucanase 2; D002480:Cellulase; D014357:Trypsin