INHIBITION OF CELLOBIOHYDROLASE-I FROM TRICHODERMA-REESEI BY PALLADIUM

Cellulase from Trichoderma reesei is a multienzyme mixture that hydrol yzes cellulose to glucose. Two enzymes in this mixture, cellobiohydrol ase (CBH) and endoglucanase (EG), possess a common structure comprisin g a distinct cellulose-binding domain (CBD) and catalytic domain. Inhi bition of the catalytic domain of cellulases without affecting their C BD function might be useful for structure/function studies of these en zymes, Complexes of the platinum group metals were tested for their ab ility to inhibit the major cellulase enzyme from T. reesei, cellobiohy drolase I (CBH I). Only palladium complexes inhibited CBH I, inhibitio n being dependent upon the molar ratio of palladium to CEH I with 1 mu M CBH I retaining only 10% of its activity in the presence of 100 mu M ammonium hexachloropalladate(IV) and after the incorporation of 28 m ol Pd/mol CBH I. Inhibition was irreversible and could be completely p revented by including histidine, cysteine, and cystine in the assay mi xture, Although the primary mechanism of inhibition of CBH I by pallad ium remains to be elucidated, it could involve the binding of palladiu m to sulfur or cystine residues resulting in their degradation, This i s based on the findings that (i) palladium-inhibited CBH I was less th ermally stable than native CBH I; (ii) CBH I, chemically modified by t he attachment of pentaammine ruthenium(III) to the imidazole-N of eith er H206 or H228, showed greater sensitivity to inhibition by palladium compared to native CBH I; and (iii) ammonium hexachloropalladate clea ved 5,5'-dithiobis(2-nitrobenzoic acid)-Ellman's reagent. Binding of C BH I to crystalline cotton linters was not affected by palladium. (C) 1995 Academic Press, Inc.