Molecular recognition in the product site of cellobiohydrolase Cel7A regulates processive step length

Johan Pelck Olsen, Jeppe Kari, Michael Skovbo Windahl, Kim Borch, Peter Westh*

*Corresponding author

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review


Cellobiohydrolase Cel7A is an industrial important enzyme that breaks down cellulose by a complex processive mechanism. The enzyme threads the reducing end of a cellulose strand into its tunnel-shaped catalytic domain and progresses along the strand while sequentially releasing the disaccharide cellobiose. While some molecular details of this intricate process have emerged, general structure-function relationships for Cel7A remain poorly elucidated. One interesting aspect is the occurrence of particularly strong ligand interactions in the product binding site. In this work, we analyze these interactions in Cel7A from Trichoderma reesei with special emphasis on the Arg251 and Arg394 residues. We made extensive biochemical characterization of enzymes that were mutated in these two positions and showed that the arginine residues contributed strongly to product binding. Specifically, ∼50% of the total standard free energy of product binding could be ascribed to four hydrogen bonds to Arg251 and Arg394, which had previously been identified in crystal structures. Mutation of either Arg251 or Arg394 lowered production inhibition of Cel7A, but at the same time altered the enzyme product profile and resulted in ∼50% reduction in both processivity and hydrolytic activity. The position of the two arginine residues closely matches the two-fold screw axis symmetry of the substrate, and this energetically favors the productive enzyme-substrate complex. Our results indicate that the strong and specific ligand interactions of Arg251 and Arg394 provide a simple proofreading system that controls the step length during consecutive hydrolysis and minimizes dead time associated with transient, non-productive complexes.
TidsskriftBiochemical Journal
Udgave nummer1
Sider (fra-til)99-110
Antal sider12
StatusUdgivet - 8 jan. 2020
Udgivet eksterntJa

Citer dette