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 X100 and beta-mercaptoethanol to the buffer yielded an active enzyme that could be stored for several months at -80 degrees 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 alpha-linolenic acid (alpha-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 microM a typical Michaelis-Menten curve was found with a K(m) of 77 microM. At higher alpha-13-HPOT concentrations inhibition of the enzyme was observed, which could (at least in part) be attributed to 2E-hexenal. A curve of the substrate conversion as a function of the enzyme concentration revealed that 1 nkat of enzyme activity converts 0.7 mumol alpha-13-HPOT before inactivation. Headspace analysis showed that tomato HPO-lyase formed hexanal from 13-HPOD and 3Z-hexenal from alpha-13-HPOT. A trace of the latter compound was isomerized to 2E-hexenal. In addition to the aldehydes, 12-oxo-9Z-dodecenoic acid was found by GC/MS analysis. To a small extent, isomerization to 12-oxo-10E-dodecenoic acid occurred.
Suurmeijer
C N
CN
Bijvoet Center, Department of Bio-organic Chemistry, Utrecht University, The Netherlands.
Pérez-Gilabert
M
M
van Unen
D J
DJ
van der Hijden
H T
HT
Veldink
G A
GA
Vliegenthart
J F
JF
D000446:Aldehyde-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System; D004795:Enzyme Stability; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*
D003577:Cytochrome P-450 Enzyme System; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase