Substrate binding in the processive cellulase Cel7A: Transition state of complexation and roles of conserved tryptophan residues.

ABSTRACT: Cellobiohydrolases effectively degrade cellulose and are of biotechnological interest because they can convert lignocellulosic biomass to fermentable sugars. Here, we implemented a fluorescence-based method for real-time measurements of complexation and decomplexation of the processive cellulase Cel7A and its insoluble substrate, cellulose. The method enabled detailed kinetic and thermodynamic analyses of ligand binding in a heterogeneous system. We studied WT Cel7A and several variants in which one or two of four highly conserved Trp residues in the binding tunnel had been replaced with Ala. WT Cel7A had on/off-rate constants of 1 × 10⁵ m^-1 s^-1 and 5 × 10^-3 s^-1, respectively, reflecting the slow dynamics of a solid, polymeric ligand.

SUBMITTER: Rojel N 

PROVIDER: S-EPMC7008363 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature