Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 287, Issue 22, Pages 18451-18458Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M111.334946
Keywords
-
Categories
Funding
- Danish Agency for Science, Technology and Innovation
- Ministry of Education, Culture, Sports, Science and Technology of Japan [23760746]
- Programme Commission on Sustainable Energy and Environment [2104-07-0028]
- Grants-in-Aid for Scientific Research [23760746] Funding Source: KAKEN
Ask authors/readers for more resources
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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available