The Lipoxygenases

The biosynthesis of the leukotrienes involves a series of reactions initiated by 5-lipoxygenase, which catalyzes both the oxygenation of arachidonate to 5(S)-HPETE and the subsequent dehydration of this hydroperoxide intermediate to produce the epoxide, 5(S),6(S)-oxido-7,9,l 1,15(E,E,Z,Z)-eicosatetraenoic 

How can one inhibit leukotriene biosynthesis From what we have seen above, there are several possible ways (Fig. 4.20). Inhibition of 5-lipoxygenase activity is the most obvious one, and it has been extensively explored. In this line, the design of drugs that interact with the iron present at the active site of 5-lipoxygenase has been particularly fruitful. To this group of compounds 

Different isoforms of 15-lipoxygenase have been purified to homogeneity from mammalian sources, including reticulocytes and leukocytes, and complementary DNAs encoding them have been cloned. The reticulocyte 15-lipoxygenase is a cytosolic protein, whose amino acid sequence is 65% similar to human 5-lipoxygenase and 45% similar to type I soybean lipoxygenase (whose tridimensional structure has been resolved by 

X-ray crystallography). Products of arachidonate metabolism via the 15-lipoxygenase pathway have been involved in hyperalgesia and inflammation, while the 15-lipoxygenase-catalyzed oxygenation of es-terified fatty acids in phospholipids and in low-density lipoproteins has been implicated in the maturation of reticulocytes to erythrocytes and in the etiology of atherosclerosis (see, for review, ref. 145). 

15-Lipoxygenase metabolites of linoleic acid (18 2 A ) are produced in tissues and may participate in cellular signaling. For example, biosynthesis of 13(S)-hydroxyoctadecadienoic acid (13-HODE) in Syrian hamster embryo fibroblasts is increased when the cells are stimulated with epidermal growth factor (EGF). Moreover, the addition of 13-HODE to the fibroblasts enhances EGF-induced proliferation.  

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There are three groups of eicosanoids that are synthesized from C20 eicosanoic acids derived from the essential fatty acids linoleate and a-linolenate, or directly from dietary arachidonate and eicosapentaenoate (Figure 23-5). Arachidonate, usually derived from the 2 position of phospholipids in the plasma membrane by the action of phospholipase Aj (Figure 24-6)—but also from the diet—is the substrate for the synthesis of the PG2, 1X2 series (prostanoids) by the cyclooxygenase pathway, or the LT4 and LX4 series by the lipoxygenase pathway, with the two pathways competing for the arachidonate substrate (Figure 23-5). 

Figure 23-7. Conversion of arachidonicacid to leukotrienesand lipoxins of series 4 via the lipoxygenase pathway. Some similar conversions occur in series 3 and 5 leukotrienes. (HPETE, hydroperoxyeicosatetraenoate HETE, hydroxyeicosatetraenoate , peroxidase (2), leukotriene A4 epoxide hydrolase , glutathione S-transferase ...

The biological membranes that surround cells and form the boundaries of intracellular organelles contain polyunsaturated fiitty acids, which are susceptible to oxidation. This reaction is used under controlled conditions by enzymes, such as the lipoxygenases or cyclooxygenases, within cells to produce oxygenated lipids, which can act as mediators of inflammation (Smith and Marnett, 1991 Yamamoto, 1992). Such compounds are characterized by their high potency and specificity in their interaction with cells (Salmon, 1986). While these enzymatic reactions... 

On the other hand, biocatalyst stability can be affected by the presence of organic-aqueous interface. In our previous work [25], we studied the effect of interface with octane on the lipoxygenase stability in an octane-buffer pH 9.6 biphasic system. This loss in activity is more pronounced than that observed in the aqueous system. During lipoxygena-tion, the active enzyme concentration [E] in the aqueous phase of our biphasic bioreactor was ... 

The lipase-catalyzed fatty acid ester hydrolysis and the lipoxygenation of free polyunsaturated fatty acids are involved in the same lipid degradation pathway. They are respectively the first and second reaction in the lipoxygenase pathway (Fig. 3) [87-91]. The pathway produces volatile products of considerable importance in food technology including Cg[92, 93] or Cg- 94—96 aldehydes and alcohols from polyunsaturated fatty... 

The kinetics of the lipoxygenase was studied in aqueous media [25]. The affinity of the enzyme for its substrate LA is very high. When LA is in excess in the medium it becomes the inhibitor of the reaction [Fig. 5(a)]. HP is also competitive inhibitor. The experimentally determined kinetic constants are ... 

Furthermore, in the system with coupled lipase and lipoxygenase, the production rate of HP is governed by the first enzymatic reaction and mass transfer. When TL,- is small (0 to 1 mM equiv. 3 mM LA), the kinetic curve has a sigmoid shape due to surface active properties of LA and HP [25]. Hydrolysis of TL and the increase of LA favor the transfer of LA. Such a transfer allows the lipoxygenase reaction to progress. Since lipox-ygenation consumes LA and produces HP, catalysis and transfer demonstrates a reciprocal influence. 

At the end of the reaction, hydroperoxide can be easily recovered in the aqueous phase (98-99%) after its separation from the organic phase and precipitation of the enzymes. The hydroperoxides obtained are highly reactive molecules [109]. They are intermediate compounds in the lipoxygenase pathway in plants, precursors for the synthesis of hydroxy-fatty acids (i.e., ( + )-coriolic acid [38,110], and regulators of the prostaglandins biosynthesis [111-113]. 

Current research is in progress in our laboratory on biocatalysis in two-phase media with j6-glycosidases, yeast, and enzymes of the lipoxygenase pathway. 

Feussner I and Wasternack C. 2002. The lipoxygenase pathway. Annu Rev Plant Physiol Plant Mol Biol... 

Flavor is one of the major characteristics that restricts the use of legume flours and proteins in foods. Processing of soybeans, peas and other legumes often results in a wide variety of volatile compounds that contribute flavor notes, such as grassy, beany and rancid flavors. Many of the objectionable flavors come from oxidative deterioration of the unsaturated lipids. The lipoxygenase-catalyzed conversion of unsaturated fatty acids to hydroperoxides, followed by their degradation to volatile and non-volatile compounds, has been identified as one of the important sources of flavor and aroma components of fruits and vegetables. An enzyme-active system, such as raw pea flour, may have most of the necessary enzymes to produce short chain carbonyl compounds. 

Figure 1.9. Overview of the biosynthesis of ecosanoids. The 20 carbon fatty acid arachidonic acid is released from cell membrane phospholipids by the actions of phospholipase A2. Free arachidonic acid forms the precursor of prostaglandins and thromboxanes via the multi-enz5une cyclooxygenase pathway, while leukotrienes are formed via the lipoxygenase pathway...

The eicosanoids, so called because of their derivation from a 20-carbon unsaturated fatty acid, arachidonic acid (eicosatetraenoic acid), are obtained from membrane phospholipids and synthesized de novo at the time of cellular stimulation. Arachidonic acid is cleaved from membrane-bound phosphatidylcholine by the enzyme phospholipase A2. Alternatively, arachidonic acid may be derived by the sequential actions of phospholipase C and diacylglyceryl lipase. Arachidonic acid can then follow either of two enzymatic pathways that result in the production of inflammatory mediators. The pathway initiated by cyclooxygenase (COX) produces prostaglandins the lipoxygenase pathway generates leukotrienes (Fig. 36.2). 

Feussner, L, Wasternaek, C. (2002). The lipoxygenase pathway. Anna. Rev. Plant Biol, 53,215-291. 

A spectrophotometric assay procedure was used to measure the lipoxygenase isoenzyme activities of fresh and stored soya containing bread improver pastes and powders (77). 

Scheme 7.2 Pathway for the enzymatic degradation of linoleic acid and linolenic acid via the lipoxygenase (LOX) pathway to Ce key aroma compounds in fruits and vegetables responsible for green notes. HPL hydroperoxide lyase, ADH alcohol dehydrogenase...

The fatty acid-like leukotrienes derived from the addition of glutathione to products of the lipoxygenase branch of the arachidonic cascade are closely associated with manifestations of asthma. Many compounds designed to antagonize leukotrienes at the receptor level incorporate long alkyl chains to mimic the leukotrienes backbone in addition to the sulfur-containing moieties that stand in for glutathione. The reaction... 

Iron-containing superoxide dismutases are present in many species of bacteria (Hassan and Fridovitch, 1978). These nonheme iron proteins have a characteristic set of EPR lines split about g = 4.2 in the ferric state, arising from the middle Kramers doublet of a rhombic high-spin site. Ferrous iron superoxide dismutase forms an S = I complex with NO that resembles the lipoxygenase-NO adduct by EPR criteria (I. Fridovich, T. Kirby, and J. C. Salerno, (1978) unpublished observations). 

NSAIDs usually do not inhibit lipoxygenase activity at concentrations that inhibit COX activity. In fact, by preventing arachidonic acid conversion via the COX pathway, NSAIDs may cause more substrate to be metabolized through the lipoxygenase pathways, leading to an increased formation of the inflammatory leukotrienes. Even among the COX-dependent pathways, inhibiting the... 

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