Studies of the cellulolytic system of Trichoderma reesei QM 9414. Analysis of domain function in two cellobiohydrolases by limited proteolysis.
Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II, 58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel). By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH II core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from Trichoderma reesei [Teeri et al. (1987) Gene 51, 43-52]. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH II in terms of adsorption and catalytic properties. Distinct functions can be attributed to the terminal peptides: for intact CBH II the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH II synergism experiments.
Tomme
P
P
Laboratory for Biochemistry, State University, Ghent, Belgium.
Van Tilbeurgh
H
H
Pettersson
G
G
Van Damme
J
J
Vandekerckhove
J
J
Knowles
J
J
Teeri
T
T
Claeyssens
M
M
D000595:Amino Acid Sequence; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D006026:Glycoside Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D008970:Molecular Weight; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D014242:Trichoderma / Q000201:enzymology*
D010446:Peptide Fragments; D002482:Cellulose; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase; D010206:Papain