Dynamic interaction of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during hydrolysis.
The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two Trichoderma reesei cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.
Palonen
H
H
VTT Biotechnology and Food Research, FIN-02044 Espoo, Finland.
Tenkanen
M
M
Linder
M
M
D002480:Cellulase / Q000737:chemistry / Q000378:metabolism*; D002482:Cellulose / Q000737:chemistry / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D014242:Trichoderma / Q000201:enzymology*
D007527:Isoenzymes; D002482:Cellulose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase