NDLI: Processivity of Cellobiohydrolases Is Limited by the Substrate

Content Provider	Journal of Biological Chemistry (JBC)
Author	Kurašin, Mihhail Väljamäe, Priit
Abstract	Processive cellobiohydrolases (CBHs) are the key components of fungal cellulase systems. Despite the wealth of structural data confirming the processive mode of action, little quantitative information on the processivity of CBHs is available. Here, we developed a method for measuring cellulase processivity. Sensitive fluorescence detection of enzyme-generated insoluble reducing groups on cellulose after labeling with diaminopyridine enabled quantification of the number of reducing-end exo-mode and endo-mode initiations. Both CBHs TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium employed reducing-end exo- and endo-mode initiation in parallel. Processivity values measured for TrCel7A and PcCel7D on cellulose hydrolysis were more than an order of magnitude lower than the values of intrinsic processivity that were found from the ratio of catalytic constant (kcat) and dissociation rate constant (koff). We propose that the length of the obstacle-free path available for a processive run on cellulose chain limits the processivity of CBHs on cellulose. TrCel7A and PcCel7D differed in their koff values, whereas the kcat values were similar. Furthermore, the koff values for endoglucanases (EGs) were much higher than the koff values for CBHs, whereas the kcat values for EGs and CBHs were within the same order of magnitude. These results suggest that the value of koff may be the primary target for the selection of cellulases.
Related Links	http://www.jbc.org/content/286/1/169.abstract
Ending Page	177
Starting Page	169
Page Count	9
File Format	HTM / HTML PDF
ISSN	00219258
Journal	Journal of Biological Chemistry (JBC)
Issue Number	1
Volume Number	286
DOI	10.1074/jbc.M110.161059
e-ISSN	1083351X
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2011-01-07
Access Restriction	Open
Subject Keyword	Carbohydrate-binding Protein Carbohydrate Chemistry Enzyme Catalysis Enzyme Kinetics Enzyme Mechanisms Catalytic Constant Cellulase Cellulose Dissociation Rate Constant Processivity Enzymology
Content Type	Text
Resource Type	Article
Subject	Cell Biology Biochemistry Molecular Biology

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