Comparison of kinetic and molecular properties of two forms of amygdalin hydrolase from black cherry (Prunus serotina Ehrh.) seeds.
Two forms of the beta-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 (Mr 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 alpha-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-beta-D-glucoside and 4-methylumbelliferyl-beta-D-glucoside but were inactive towards (R)-prunasin, p-nitrophenyl-alpha-D-glucoside, and 4-methylumbelliferyl-alpha-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 delta-gluconolactone and castanospermine.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005638:Fruit / Q000201:enzymology*; D005959:Glucosidases / Q000302:isolation & purification*; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000302:isolation & purification* / Q000378:metabolism
D007527:Isoenzymes; D005959:Glucosidases; C032638:amygdalin beta(1-6)glucosidase; D001617:beta-Glucosidase