title: Enhancement of the affinity of cellobiohydrolase I and its catalytic domain to cellulose in the presence of the reaction product--cellobiose.
abstract: The catalytic domain of cellobiohydrolase I from Trichoderma reesei has been obtained by papain treatment of the native enzyme adsorbed onto the surface of microcrystalline cellulose. Both the intact and the truncated enzyme are almost equally active toward soluble fluorogenic derivatives of cellobi-, -tri-, -tetra-, and -pentaose, the fastest and the slowest fluorophore liberation being observed for MUF-cellopenta- and -tetraose, respectively. Titration of the active centers of the intact enzyme and its catalytic domain with MUF-cellotetraose showed their molecular masses to be 49 and 39 kD, respectively, the dissociation constants of the enzyme-soluble ligand complexes being almost equal (65 and 70 nM at 20 degrees C, respectively). In contrast, the intact enzyme and its catalytic core have been shown to significantly (50-60 times) differ in their affinity to insoluble microcrystalline cellulose at low enzyme loading (up to 10 mg per g of the substrate). At 20 degrees C the dissociation constants for the two forms of the enzyme are estimated to be 10 and 500 nM, respectively. Surprisingly, under these conditions the reaction product and inhibitor, cellobiose (Ki = 10 microM), at the concentration 10 mM, increased 3-4-fold the affinity of both the intact cellobiohydrolase and its catalytic domain to cellulose.
authors.affiliation: Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 117071, Russia. inbio@glas.apc.org
authors.forename: M L
authors.initials: ML
authors.lastname: Herner
authors.forename: M S
authors.initials: MS
authors.lastname: Melnick
authors.forename: M L
authors.initials: ML
authors.lastname: Rabinovich
mesh_terms: D002475:Cellobiose / Q000494:pharmacology*; D002480:Cellulase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D002475:Cellobiose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase