Protein Engineering vol. 9 no. 8 pp. 691-699, 1996
© 1996 Oxford University Press
RESEARCH-ARTICLE |
The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169
VTT Biotechnology and Food Research PO Box 1500, FIN-02044 VTT, Espoo, Finland 4Laboratory of Biochemistry, University of Ghent K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium 5Department of Molecular Biology BMC, PO Box 590, S-75124 Uppsala, Sweden
1Author to whom correspondence should be addressed
Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The ß-l,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified 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 behaviour of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 Å 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 increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have asecond role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.
Keywords: catalytic domain/cellobiohydrolase/site-directed/mutgenesis/Trichoderma reesei
Received January 2, 1996; revised April 10, 1996; accepted April 18, 1996.
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