THE ACTIVE-SITE OF TRICHODERMA-REESEI CELLOBIOHYDROLASE-II - THE ROLEOF TYROSINE-169

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cle aving primarily cellobiose units from the non-reducing end of cellulos e chains, The beta-1,4 glycosidic bond is cleaved by acid catalysis wi th an aspartic acid, D221, as the likely proton donor, and another asp artate, D175, probably ensuring its protonation and stabilizing charge d reaction intermediates, The catalytic base has not yet been identifi ed experimentally, The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis, The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding b ehaviour of the mutated CBHII, The crystal structure of the mutated en zyme was determined to 2.0 Angstrom resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are inc reased threefold and for 4-methylumbelliferyl cellobioside over 50-fol d. The catalytic constants towards cellotriose and cellotetraose are f our times lower for the mutant, These data suggest that Y169, on inter acting with a glucose ring entering the second subsite in a narrow tun nel, helps to distort the glucose ring into a more reactive conformati on, In addition, a change in the pH activity profile was observed, Thi s indicates that Y169 may have a second role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.