Comparison of kinetic and molecular properties of two forms of amygdalin hydrolase from black cherry ( Prunus serotina Ehrh.) seeds

Abstract

Two forms of the β-glucosidase amygdalin hydrolase (AH I and II), which catalyze the hydrolysis of ( R)-amygdalin to ( R)-prunasin and d-glucose, have been purified over 200-fold from mature black cherry ( Prunus serotina Ehrh.) seeds. These proteins showed very similar molecular and kinetic properties but could be resolved by chromatofocusing and isoelectric focusing. AH I and II were monomeric ( M r 60,000) and had isoelectric points of 6.6 and 6.5, respectively. Their glycoprotein character was indicated by positive periodic acid-Schiff staining and by their binding to concanavalin A-Sepharose 4B with subsequent elution by α-Me- d-glucoside. Of the natural glycosidic substrates tested, both enzymes showed a pronounced preference for the endogenous cyanogenic disaccharide ( R)-amygdalin. They also hydrolyzed at the same active site the synthetic substrates p-nitrophenyl-β- d-glucoside and 4-methylumbelliferyl-β- d-glucoside but were inactive towards ( R)-prunasin, p-nitrophenyl-α- d-glucoside, and 4-methylumbelliferyl-α- d-glucoside. Maximum hydrolytic activity was shown in citrate-phosphate buffer in the pH range 4.5–5.0. AH I and II were inhibited competitively by the reaction product ( R)-prunasin and noncompetitively (mixed type) by Δ-gluconolactone and castanospermine.