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

被引:95
作者
Cruys-Bagger, Nicolaj [1 ]
Elmerdahl, Jens [1 ]
Praestgaard, Eigil [1 ]
Tatsumi, Hirosuke [2 ]
Spodsberg, Nikolaj [3 ]
Borch, Kim [3 ]
Westh, Peter [1 ]
机构
[1] Roskilde Univ, Dept Sci Syst & Models, Res Unit Funct Biomat, DK-4000 Roskilde, Denmark
[2] Shinshu Univ, Int Young Researchers Empowerment Ctr, Matsumoto, Nagano 3908621, Japan
[3] Novozymes AS, DK-2880 Bagsvaerd, Denmark
关键词
TRICHODERMA-REESEI; ENZYMATIC-HYDROLYSIS; CELLOBIOSE DEHYDROGENASE; PHANEROCHAETE-CHRYSOSPORIUM; MICROCRYSTALLINE CELLULOSE; CRYSTALLINE CELLULOSE; CELLULASES; SUBSTRATE; ADSORPTION; SYNERGISM;
D O I
10.1074/jbc.M111.334946
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 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.
引用
收藏
页码:18451 / 18458
页数:8
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