Isolation and characterization of multiple forms of mandelonitrile lyase from mature black cherry (Prunus serotina Ehrh.) seeds.
Five multiple forms (forms 1-5) of mandelonitrile lyase (EC 4.1.2.10) which catalyze the decomposition of mandelonitrile to benzaldehyde and hydrogen cyanide have been extensively purified from seeds of black cherry (Prunus serotina Ehrh.) by concanavalin A-Sepharose 4B chromatography and chromatofocusing. These forms are monomers which differ only slightly in molecular weight (57,000-59,000) and isoelectric point (4.58-4.63), but heterogeneity in their carbohydrate side-chains was suggested by concanavalin A-Sepharose 4B chromatography. The absorption spectra of the predominating forms 4 and 5 showed maxima of 278, 380, and 460 nm, indicative of flavoprotein character. Detailed comparative kinetic studies of forms 4 and 5 revealed few significant differences in behavior. Both proteins showed pH optima between 6.0 and 7.0, had identical Km values (0.17 mM) for (R,S)-mandelonitrile, and retained similar activities upon storage at 4 and -20 degrees C. Neither form exhibited a metal ion requirement and both were affected similarly by metal salts, beta-mercaptoethanol, and sulfhydryl reagents. Benzoic acid, p-hydroxybenzyl alcohol, and benzyl alcohol inhibited both forms.
D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002021:Buffers; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography / Q000379:methods; D005638:Fruit / Q000201:enzymology*; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D012639:Seeds / Q000201:enzymology*
D002021:Buffers; D007527:Isoenzymes; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase