Purification, stabilization and characterization of tomato fatty acid hydroperoxide lyase

Fatty acid hydroperoxide lyase (HPO-lyase) was purified 300-fold from tomatoes. The enzymatic activity appeared to be very unstable, but addition of Triton X 100 and β-mercaptoethanol to the buffer yielded an active enzyme that could be stored for several months at −80°C. The enzyme was inhibited by desferoxamine mesylate (desferal), 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone), nordihydroguaiaretic acid (NDGA), n-propyl gallate and butylated hydroxyanisole, suggesting the involvement of free radicals in the reaction mechanism and the existence of a prosthetic group in the active center. However, no heme group could be demonstrated with the methods commonly used to identify heme groups in proteins. Only 13-hydroperoxides from linoleic acid (13-HPOD) and α-linolenic acid (α-13-HPOT) were cleaved by the tomato enzyme, with a clear preference for the latter substrate. The pH-optimum was 6.5, and for concentrations lower than 300 μM a typical Michaelis–Menten curve was found with a K m of 77 μM. At higher α-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2 E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 μmol α-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3 Z-hexenal from α-13-HPOT. A trace of the latter compound was isomerized to 2 E-hexenal. In addition to the aldehydes, 12-oxo-9 Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10 E-dodecenoic acid occurred.