Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Nicolaj Cruys-Bagger, Jens Elmerdahl Olsen, Eigil Præstgaard, Hirosuke Tatsumi, Nikolaj Spodsberg, Kim Borch, Peter Westh

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

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.
OriginalsprogEngelsk
TidsskriftJournal of Biological Chemistry
Vol/bind287
Udgave nummer22
Sider (fra-til)18451-18458
ISSN0021-9258
DOI
StatusUdgivet - 2012

Citer dette

Cruys-Bagger, N., Olsen, J. E., Præstgaard, E., Tatsumi, H., Spodsberg, N., Borch, K., & Westh, P. (2012). Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A. Journal of Biological Chemistry, 287(22), 18451-18458. https://doi.org/10.1074/jbc.M111.334946