Front Matter

chanistic data available, and it is rather difficult to prove or disprove either of these

models experimentally. A key intermediate in the radical mechanism, the carbon-

centered alkyl radical has apparently been detected by EPR (Pistorius et al.,

1976). In contrast, there is no direct experimental evidence for the formation of

the organoiron intermediate. However, experimental data are available which sug-

gest the formation of organoiron complexes between LOXs and hydroperoxy fatty

acids (Nelson et al., 1994).

In addition to the naturally occurring polyenoic fatty acids containing (1Z,4Z)-

pentadienyl systems, LOXs may also oxygenate unusual substrates such as allylic

ketones. For instance, the keto derivative of ricinolic acid may be metabolized into a

conjugated unsaturated diketo fatty acid or may be cleaved into the correspondingx-

keto C13–fatty acid (Ku ̈hn et al., 1991a). Furthermore, oxygenated polyenoic fatty

acids which still contain doubly allylic methylenes such as 12- or 15-HETE can be

converted to double-oxygenation products (Bild et al., 1977; van Os et al., 1981;

Schwarz et al., 1998).

15.2.2 Hydroperoxidase activity

Under certain reaction conditions such as reduced oxygen pressure and in the pre-

sence of a reductant, which reduces the ferric enzyme back to its ferrous state, LOX

may catalyze the degradation of hydroperoxy lipids to an array of secondary pro-

ducts. Polyenoic fatty acids are preferred as reductant, but other hydrophobic redu-

cing agents such as guaiacol may also be used. The hydroperoxidase reaction is

initiated by a homolytic cleavage of the peroxy bond forming alkoxy and hydro-

xyl-radicals. These radical intermediate may react with other components in the

assay system so that a variety of secondary products may be formed (Figure 1, anae-

robic cycle) (Garssen et al., 1971; De Groot et al., 1973; Ku ̈hn et al., 1991b).

15.2.3 Leukotriene synthase activity

Hydroperoxy fatty acids which still contain bisallylic methylenes can be converted

by LOXs to epoxy leukotriene derivatives. For instance, 5-HPETE can be converted

by purified 5/8-LOXs to 5,6-epoxy leukotriene A 4 ((5S,6S,7E,9E,11Z)-5,6-epoxy-

7,9,11-eicosatrienoic acid) (Shimizu et al., 1984). Similarly, 15-HPETE may be

transformed by 12/15-LOXs to the corresponding 14,15-leukotriene A 4 (Bryant

et al., 1985). The leukotriene synthase activity of LOXs may be regarded as com-

bination of oxygenase and hydroperoxidase activity. Hydroperoxy fatty acids which

still contain at least one bisallylic methylene are substrates for leukotriene formation

and the reaction involves both, stereoselective removal of a hydrogen from a bis-

allylic methylene and homolytic cleavage of the hydroperoxy group. The biradical

formed via these reactions is stabilized during epoxide formation. Epoxy leuko-

trienes are rather unstable compounds and rapidly undergo epoxide hydrolysis at

acidic pH. The resulting diols are frequently used as indicators for the leukotriene

synthase activity of LOXs.

312 15 Application of Lipoxygenases and Related Enzymes