title: Rate of Threading a Cellulose Chain into the Binding Tunnel of a Cellulase.
abstract: Industrially important cellulase Cel7A hydrolyzes crystalline cellulose by a complex processive mechanism in which the enzyme slides along the cellulose surface with one strand of the polymeric substrate channeled through its catalytic tunnel. Each processive run must start with threading the tunnel with a cellulose strand and end with the opposite, that is, the dethreading process. Evidence has suggested that threading or dethreading may be rate-limiting for the overall enzyme reaction. To directly elucidate the rates of threading and dethreading, we analyzed experimental data with respect to a two-step model that distinguishes enzymes in free, associated nonthreaded, and threaded states. This approach enabled the estimation of rate constants for both steps in both directions. The results showed that Cel7A utilizes a "tapping" mode of attack, in which it associates unproductively with the cellulose surface many times before it eventually finds a location at which it gets threaded. Moreover, it was concluded that at the quasi steady state dethreading was the main determinant of the overall hydrolytic rate under most conditions. An exception to this was at very low enzyme/substrate ratios, at which other steps also influenced the overall dynamics. These results will be helpful in identifying rate-limiting steps for cellulases and, in turn, targets for rational design of faster enzymes.
authors.affiliation: Department of Science and Environment, Roskilde University , 1 Universitetsvej, DK-4000 Roskilde, Denmark.|Novozymes A/S , Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.affiliation: Department of Science and Environment, Roskilde University , 1 Universitetsvej, DK-4000 Roskilde, Denmark.
authors.forename: Kadri
authors.initials: K
authors.lastname: Alasepp
authors.affiliation: Department of Science and Environment, Roskilde University , 1 Universitetsvej, DK-4000 Roskilde, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Andersen
authors.affiliation: Department of Science and Environment, Roskilde University , 1 Universitetsvej, DK-4000 Roskilde, Denmark.
authors.forename: Johnny
authors.initials: J
authors.lastname: Ottesen
authors.affiliation: Novozymes A/S , Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.affiliation: Department of Science and Environment, Roskilde University , 1 Universitetsvej, DK-4000 Roskilde, Denmark.
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D020134:Catalytic Domain; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D014242:Trichoderma / Q000201:enzymology
chemical_list: D005656:Fungal Proteins; D002482:Cellulose; D002480:Cellulase