Purification of the alliin lyase of garlic, Allium sativum L.
1. Alliin lyase (EC 4.4.1.4) was purified up to sevenfold from garlic-bulb homogenates. The enzyme was unstable to storage at -10 degrees , particularly in dilute concentrations, but the addition of glycerol (final concentration 10%, v/v) stabilized the activity completely for at least 30 days. 2. The purified enzyme had an optimum pH for activity at 6.5. The addition of pyridoxal phosphate stimulated the reaction rate and the stimulation became more marked as the purification proceeded. 3. Hydroxylamine (10mum) and cysteine (0.5mm) inhibited the enzyme activity by more than 80%. Spectral studies indicated that cysteine reacted with pyridoxal phosphate bound to the protein. 4. The K(m) values for S-methyl-, S-ethyl-, S-propyl-, S-butyl- and S-allyl-l-cysteine sulphoxides were determined. With S-allyl-l-cysteine sulphoxide the K(m) was 6mm and the V(max.) was greater than those with the other substrates tested. 5. The thioether analogues of the substrates were competitive inhibitors for the lyase reaction. The K(i) decreased with increasing chain length of the alkyl substituent. With S-ethyl-l-cysteine sulphoxide as substrate the K(i) was 33, 8 and 5mm respectively for S-methyl-, S-ethyl- and S-propyl-l-cysteine. 6. The addition of EDTA or Mg(2+), Mn(2+), Co(2+) or Fe(2+) stimulated the reaction rate. Other bivalent cations either had no effect or gave a strong inhibition. In the presence of EDTA no further increase of activity was observed with added Mg(2+).
D055598:Chemical Phenomena; D002621:Chemistry; D002850:Chromatography, Gel; D003035:Cobalt; D003080:Cold Temperature; D003545:Cysteine; D004355:Drug Stability; D004492:Edetic Acid; D005737:Garlic / Q000201:enzymology*; D005990:Glycerol; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines; D007501:Iron; D007700:Kinetics; D008190:Lyases / Q000037:antagonists & inhibitors; D008274:Magnesium; D008345:Manganese; D010946:Plants, Medicinal; D011732:Pyridoxal Phosphate; D013053:Spectrophotometry; D013454:Sulfoxides; D013997:Time Factors
D006898:Hydroxylamines; D013454:Sulfoxides; D003035:Cobalt; D008345:Manganese; D011732:Pyridoxal Phosphate; D004492:Edetic Acid; D007501:Iron; D008190:Lyases; D008274:Magnesium; D003545:Cysteine; D005990:Glycerol