Kinetic investigation of the substrate specificity of the cyanogenic β-d-glucosidase (linamarase) of white clover

Partially purified linamarase from Trifolium repens (genotype Lili acac ) plants was kinetically characterized. Kinetic evidence was found to support the assumption that this cyanogenic β- d -glucosidase has a broad substrate spectrum. p -Nitrophenyl-β- d -xylopyranoside and p -nitrophenyl-α- l -arabinopyranoside substrates bound almost as tightly to the active center of the enzyme as the glucono(1 → 5)lactone transition-state analog inhibitor. Substrate specificity investigation also indicated that positions C-4 and C-6 on the pyranoside ring play an essential role in both substrate orientation and splitting. Recently very similar kinetic characteristics were reported on some mammalian cytosolic β- d -glucosidases and a possible physiological interpretation of this coincidence is discussed. Inhibition studies with glucono(1 → 5)lactone revealed that the carbohydrate moiety of each substrate attached to the same binding site in the active center. Inhibition experiments with 1-thio substrate analogs demonstrated that the aglycon and the angular arrangement around the glycosidic linkage were the major determinants in the observed substrate specificity.