Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

Jeppe Kari, Johan Pelck Olsen, Kim Borch, Nicolaj Cruys-Bagger, Kenneth Jensen, Peter Westh

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    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)
    TidsskriftJournal of Biological Chemistry
    Sider (fra-til)32459-32468
    StatusUdgivet - 30 sep. 2014


    • Biodegradation
    • Biofuel
    • Cellobiohydrolase
    • Cellulose
    • Enzyme Kinetics
    • Crystalline Cellulose Degradation
    • Trichoderma reesei

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