The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanasecleaving primarily cellobiose units from the non-reducing endof cellulose chains. The ß-l,4 glycosidic bond iscleaved by acid catalysis with an aspartic acid, D221, as thelikely proton donor, and another aspartate, D175, probably ensuringits protonation and stabilizing charged reaction intermediates.The catalytic base has not yet been identified experimentally.The refined crystal structure of CBHII also shows a tyrosineresidue, Y169, located close enough to the scissile bond tobe involved in catalysis. The role of this residue has beenstudied by introducing a mutation Y169F, and analysing the kineticand binding behaviour of the mutated CBHII. The crystal structureof the mutated enzyme was determined to 2.0 Å resolutionshowing no changes when compared with the structure of nativeCBHII. However, the association constants of the mutant enzymefor cellobiose and cellotriose are increased threefold and for4-methylumbelliferyl cellobioside over 50-fold. The catalyticconstants towards cellotriose and cellotetraose are four timeslower for the mutant. These data suggest that Y169, on interactingwith a glucose ring entering the second subsite in a narrowtunnel, helps to distort the glucose ring into a more reactiveconformation. In addition, a change in the pH activity profilewas observed. This indicates that Y169 may have asecond rolein the catalysis, namely to affect the protonation state ofthe active site carboxylates, D175 and D221.