Trade-off between Processivity and Hydrolytic Velocity of Cellobiohydrolases at the Surface of Crystalline Cellulose

Abstract

Analysis of heterogeneous catalysis at an interface is difficult because of the variety of reaction sites and the difficulty of observing the reaction. Enzymatic hydrolysis of cellulose by cellulases is a typical heterogeneous reaction at a solid/liquid interface, and a key parameter of such reactions on polymeric substrates is the processivity, i.e., the number of catalytic cycles that can occur without detachment of the enzyme from the substrate. In this study, we evaluated the reactions of three closely related glycoside hydrolase family 7 cellobiohydrolases from filamentous fungi at the molecular level by means of high-speed atomic force microscopy to investigate the structure−function relationship of the cellobiohydrolases on crystalline cellulose. We found that high moving velocity of enzyme molecules on the surface is associated with a high dissociation rate constant from the substrate, which means weak interaction between enzyme and substrate. Moreover, higher values of processivity were associated with more loop regions covering the subsite cleft, which may imply higher binding affinity. Loop regions covering the subsites result in stronger interaction, which decreases the velocity but increases the processivity. These results indicate that there is a trade-off between processivity and hydrolytic velocity among processive cellulases.

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

“…Measuring the processivity of glycoside hydrolases is not straightforward, and the results are dependent on the method used (18,54). The lengths of the processive runs of TrCel7A measured on different celluloses using different methods for measuring are in the range of 10 -70 (17,23,26,29,43,55,56). Clearly these figures are far lower than P Intr values of 4000 and 560 measured on bacterial cellulose and amorphous cellulose, respectively (17).…”

“…Using the global kinetic modeling of progress curves (12,26) and single molecule fluorescence imaging (28), k off values for TrCel7A in the order of 10 Ϫ2 s Ϫ1 were obtained. Using high speed atomic force microscopy, the k off values measured for TrCel7A were in the order of 10 Ϫ1 s Ϫ1 (29,30). For the cellulases of the bacterium Thermobifida fusca, the k off values between 10 Ϫ2 and 10 Ϫ3 s Ϫ1 have been measured using FRAP (31).…”

Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin

“…During our previous HS-AFM observations of processive cellulases, we realized that enzyme molecules bound nonproductively, such as those of GH family 7 endoglucanase I from T. reesei (TrCel7B), which lacks hydrolytic activity towards crystalline cellulose, are hardly visualized 18 . This is because molecules without hydrolytic activity would quickly dissociate from the substrate and therefore would not be clearly visualized in more than one frame.…”

“…Rather, we suggest that the presence of a long substrate-binding site (more than 10 GlcNAc units) formed by the ChBD is one of the key factors to high processivity of chitinases. In our recent study about fungal GH family 7 CBHs using HS-AFM, there are several loops covering subsites and these may interfere with release of the substrate from the catalytic domain, resulting in slower movement of TrCel7A than similar CBHs from basidiomycete Phanerochaete chrysosporium (PcCel7C and PcCel7D), showing trade-off relationship between hydrolytic velocity and processivity 18 . Moreover, Payne et al 25 recently demonstrated that the processivity of GH family 7 CBHs directly relates to the ligand binding free energy calculated by molecular dynamics, indicating that higher affinity to the substrate chain causes higher processivity of these enzymes.…”

Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

“…Numerous earlier studies have addressed this question, and interestingly, it appears as if the conclusions fall mainly into two mutually conflicting groups. Thus, a number of works have used either direct experimental evidence or re-examination of broader, previously published material to conclude that the overall rate is governed by the association of enzyme and substrate, particularly the placement of a cellulose strand in the long active tunnel (11)(12)(13)(14)(15)(16)(17)(18). In contrast to this, other studies, including work by this group on the pre-steady state kinetics of wild type Cel7A, have suggested that the formation of the activated complex is quite rapid and that dissociation of enzyme, which is in a position where further processive movement is hindered, is the rate-limiting step (19 -25).…”