PURIFICATION AND CHARACTERIZATION OF A C-S-LYASE FROM RAMSON, THE WILD GARLIC, ALLIUM-URSINUM

A C-S-lyase preparation from ramson, Allium ursinum L., has been purified to apparent homogeneity. Separation techniques applied were hydrophobic interaction chromatography, anion exchange chromatography, and gel permeation chromatography. A 52-fold purification was obtained. The enzyme could be characterized by a molecular mass of M(r) = 150 000 with subunits of 50 000. Its isoelectric point was determined to be at 4.7. The pH-optimum for the substrate-dependent turnover was found at 6.0. The temperature optimum was at 35 degrees C. (+)-Alliin as the substrate caused the highest enzymatic reaction velocity. The lowest K-m value was observed with (+)-S-propyl-L-cysteine sulfoxide. Inhibitor constants were elaborated for the deoxy-derivatives of the substrates inserted and, likewise, for related amino acids. The protein was sensitive to low concentrations of hydroxylamine, indicating pyridoxal phosphate as a cofactor. Activation energies were determined for the cleavage of alliin, S-propyl-L-cysteine sulfoxide and S-methyl-L-cysteine sulfoxide, and were found to be in the range of 9 to 13 kJ . mol(-1).