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)
Originalsprog | Engelsk |
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Tidsskrift | Journal of Biological Chemistry |
Vol/bind | 289 |
Sider (fra-til) | 32459-32468 |
ISSN | 0021-9258 |
DOI | |
Status | Udgivet - 30 sep. 2014 |
Emneord
- Biodegradation
- Biofuel
- Cellobiohydrolase
- Cellulose
- Enzyme Kinetics
- Crystalline Cellulose Degradation
- Trichoderma reesei