Isolation and characterization of multiple forms of prunasin hydrolase from black cherry (Prunus serotina Ehrh.) seeds.

Three forms of prunasin hydrolase (PH I, PH IIa, and PH IIb), which catalyze the hydrolysis of (R)-prunasin to mandelonitrile and D-glucose, have been purified from homogenates of mature black cherry (Prunus serotina Ehrh.) seeds. Hydroxyapatite chromatography completely resolved PH I from PH IIa and PH IIb. PH IIa and IIb, which coeluted on hydroxyapatite, were resolved by gel filtration. PH IIa was a dimer with a native molecular weight of 140,000. Both PH I and PH IIb were monomeric with molecular weights of 68,000. The isozymes appeared to be glycoproteins based on their binding to concanavalin A-Sepharose 4B with subsequent elution by alpha-methyl-D-glucoside. When presented several potential glycosidic substrates, these enzymes exhibited a narrow specificity towards (R)-prunasin. Km values for (R)-prunasin for PH I, PH IIa, and PH IIb were 1.73, 2.3, and 1.35 mM, respectively. PH I and PH IIb possessed fivefold greater Vmax/Km values than PH IIa. Ortho- and para-nitrophenyl-beta-D-glucosides were hydrolyzed at the same active site. All forms had a pH optimum of 5.0 in citrate-phosphate buffer. PH I and PH IIb were competitively inhibited by castanospermine with Ki values of 0.19 and 0.09 mM, respectively. PH activity was not stimulated by any metal ion tested and was unaffected by diethyldithiocarbamate, o-phenanthroline, 2,2'-dipyridyl, and EDTA.