title: Kinetics of cellobiohydrolase (Cel7A) variants with lowered substrate affinity.
abstract: Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for cellobiohydrolases remains controversial, and recent reports have alternately suggested either association (on-rate) or dissociation (off-rate) as the overall bottleneck. Obviously, this uncertainty hampers both fundamental mechanistic understanding and rational design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens complex formation, and the population of substrate-bound W38A was only about half of the wild type. Nevertheless, the maximal, steady-state rate was twice as high for the variant enzyme. It is argued that these opposite effects on binding and activity can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process).
authors.affiliation: From Department of Science, Systems and Models, Research Unit for Functional Biomaterials, Roskilde University, 1 Universitetsvej, DK-4000 Roskilde, Denmark and.
authors.forename: Jeppe
authors.initials: J
authors.lastname: Kari
authors.affiliation: From Department of Science, Systems and Models, Research Unit for Functional Biomaterials, Roskilde University, 1 Universitetsvej, DK-4000 Roskilde, Denmark and.
authors.forename: Johan
authors.initials: J
authors.lastname: Olsen
authors.affiliation: Novozymes A/S, Krogshøjvej 36, Bagsværd DK-2880, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.affiliation: From Department of Science, Systems and Models, Research Unit for Functional Biomaterials, Roskilde University, 1 Universitetsvej, DK-4000 Roskilde, Denmark and.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.affiliation: Novozymes A/S, Krogshøjvej 36, Bagsværd DK-2880, Denmark.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Jensen
authors.affiliation: From Department of Science, Systems and Models, Research Unit for Functional Biomaterials, Roskilde University, 1 Universitetsvej, DK-4000 Roskilde, Denmark and pwesth@ruc.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics; D002475:Cellobiose / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D002475:Cellobiose; D014364:Tryptophan; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase; D000409:Alanine
keywords: Biodegradation; Biofuel; Cellobiohydrolase; Cellulose; Crystalline Cellulose Degradation; Enzyme Kinetics; Trichoderma reesei