title: Pre-steady-state kinetics for hydrolysis of insoluble cellulose by cellobiohydrolase Cel7A.
abstract: The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10-25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ~30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.
authors.affiliation: Department of Science, Systems, and Models, Roskilde University, Roskilde, Denmark.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.forename: Jens
authors.initials: J
authors.lastname: Elmerdahl
authors.forename: Eigil
authors.initials: E
authors.lastname: Praestgaard
authors.forename: Hirosuke
authors.initials: H
authors.lastname: Tatsumi
authors.forename: Nikolaj
authors.initials: N
authors.lastname: Spodsberg
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D000327:Adsorption; D015374:Biosensing Techniques; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics
chemical_list: D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase