Cellobiohydrolases (CBHs) are exo-active glycosyl hydrolases that processively converts cellulose to soluble sugars; typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme cocktails used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for CBHs 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 enzymes with improved industrial applicability. To elucidate the role of respectively on- and off-rates on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp38) 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.
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