Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

Abstract

Background:To elucidate the rate-determining steps of cellobiohydrolase Cel7A from T. reesei, variants with lower substrate affinity were designed. Results: Mutant (W38A) had reduced substrate affinity but a 2-fold increase in the maximum quasi-steady-state rate. Conclusion: Dissociation of stalled TrCel7A is the rate-limiting step in the initial phase of hydrolysis. Significance: This work offers a new perspective for the design of faster cellulases.

Temperature Effects on Kinetic Parameters and Substrate Affinity of Cel7A Cellobiohydrolases

“…If CBM-cellulose interactions indeed contribute to a slower dissociation, higher maximal rates for the one-domain variants could reflect faster dissociation of unproductively bound enzyme (and hence earlier recruitment for new attacks). This interpretation parallels a recent study where the substrate affinity of Hj CBM was lowered by the mutation W38A in the catalytic domain (25). Kinetic analysis of this variant at 25°C showed a 4-fold increase in p K m and a 2-fold increase in p V max compared with the wild type.…”

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

“…Similar trends have also been observed with processivity-deficient variants of the other S. marcescens chitinase, ChiB (10). Studies of TrCel7A variants with lower processivity also revealed higher activity compared with the wild type enzyme on amorphous cellulose (12,43). Thus, the negative effect of processivity (slow off-rate) in hydrolysis of amorphous substrates seems to be a general feature among glycoside hydrolases evolved to degrade recalcitrant polysaccharides (7,43).…”

“…In contrast, the activity on crystalline chitin was poor, indicating the importance of processivity in the crystalline substrate degradation (7,11). The reduction of processivity upon changing Trp to Ala in substrate-binding sites has been demonstrated also for fungal (12) as well as bacterial cellulases (13)(14)(15)(16).…”

“…the experimentally measured processivity on a real polymer) is much lower than P Intr predicted from the k cat /k off ratio, indicating that the true processive ability is seldom realized. Furthermore, numerous studies have pointed out that the overall rate of processive cellulose hydrolysis is limited by the slow dissociation of the cellobiohydrolases (12,17,(21)(22)(23)(24)(25)(26). This raises the question as to why processive cellulases have evolved to such low k off values.…”

“…In case the TrCel7A-generated insoluble reducing groups on reduced cellulose under single-hit conditions were fluorescence-labeled, the k off value on the order of 10 Ϫ3 s Ϫ1 was obtained (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).…”