Substrate specificity, hydroperoxide

Major products of the lipoxygenase reaction are 9- and 13-hydroperoxides of IV and V, VI, VII, VIII and IX. Hydroperoxide lyase utilizes the 9- and/or 13-hydroperoxides (VI, VII, VIII and IX). Based on substrate specificity, hydroperoxide lyase is classified into three types. The first type is 9-hydroperoxide-specific. 

Substrate Specificity. Hydroperoxide dehydrase from sunflower cotyledons was about four-fold more active wiA 13-hydroperoxylinoleic acid Aan 13-hydroperoxylinolenic acid. In contrast, leaf hydroperoxide lyase was more Aan 10-fold more active wiA 13-hydroperoxylinolenic acid Aan wiA 13-hydroperoxylinoleic acid. However, root hydroperoxide lyase showed no preference between Ae two substrates, again suggesting Aat roots have a different form of Ae enzyme Aan leaves. NeiAer 9-hydroperoxylinoleic nor 9-hydroperoxylinolenic acids were suitable substrates for any of Ae hydroperoxide metabolizing enzymes in sunflower. 

Applications of peroxide formation are underrepresented in chiral synthetic chemistry, most likely owing to the limited stability of such intermediates. Lipoxygenases, as prototype biocatalysts for such reactions, display rather limited substrate specificity. However, interesting functionalizations at allylic positions of unsaturated fatty acids can be realized in high regio- and stereoselectivity, when the enzymatic oxidation is coupled to a chemical or enzymatic reduction process. While early work focused on derivatives of arachidonic acid chemical modifications to the carboxylate moiety are possible, provided that a sufficiently hydrophilic functionality remained. By means of this strategy, chiral diendiols are accessible after hydroperoxide reduction (Scheme 9.12) [103,104]. 

Although LOX from soybean seed is the best characterized of plant LOXs, this enzyme is present in a wide variety of plant and animal tissues (Liavonchanka and Feussner, 2006). The enzyme occurs in a variety of isoenzymes, which often vary in their optimum pH and in product and substrate specificity. Given the occurrence of multiple LOX isoenzymes in soybean leaves and the proposed roles of these enzymes in the plant metabolism, it is possible that individual isoenzymes play specific functions (Feussner and Wasternack 2002). The molecular structure of soybean LOX is the most reported, and four isoenzymes have been isolated (Baysal and Demirdoven 2007). Soy isoenzyme 1 produces 9- and 13-hydroperoxides (1 9) when the enzyme acts on free PUFA at pH 9.0, its optimum pH (Lopez-Nicolas and others 1999). Soy isoenzyme 2 acts on triglycerides as well as free PUFA leading to 9- and 13-hydroperoxide... 

An efficient method to prepare enantiomerically enriched hydroperoxides is the enzymatic kinetic resolution of racemic hydroperoxides using different kinds of enzymes (mainly lipases, chloroperoxidase, horseradish peroxidase). However, the scope of these reactions may be limit by the narrow substrate specificity of the enzyme. 

Fatty acid hydroperoxide lyase is one of the enzymes responsible for volatile Cft- and C9-aldehyde formation from linoleic and linolenic acid. This enzyme cleaves 9- and/or 13-hydroperoxides derived from linoleic and linolenic acid. The enzyme is distributed in a wide range of plant species in membrane bound forms both chloroplastic and non-chloroplastic. Three types of hydroperoxide lyases have been reported 9-hydroperoxide-specific, 13-hydroperoxide-specific and nonspecific. Other properties of the hydroperoxide lyase including substrate specificity and reaction mechanism are discussed in this review. 

The stabilizing effect of two substrate specific enzymes, superoxide dismutase (SOD) and catalase, on dHG have also been studied. Superoxide (Oo ) is the specific substrate for SOD. Hydrogen peroxide (H2O2) or short chain (i.e. CH - or C2H5-) hydroperoxides are the specific substrates for catalase. 

Although LOX from tomato fruits forms predominantly 9-hydroperoxides from linoleic and linolenic acids (Matthew et al., 1977), the cleavage enzyme from tomato does not attack these positional isomers but, rather, is specific for the 13-hydroperoxy isomers, producing hexanal or c/5-3-hexanal, respectively (Galliard and Matthew, 1977). Thus one can rationalize the formation of both Cg and C9 volatiles aldehydes in cucumber extracts with less specificity of LOX and cleavage enzymes and the absence of C9 volatiles in tomato with the substrate specificity of the cleavage enzyme. 

Although the HPLC-CL approach allows the detection of hydroperoxides at the picomole levels in oxidized lipid extracts from complex biological samples, many problems arise in recovery of different hydroperoxides, in differences in substrate specificities between CL cocktails, mistaken structural assignment for different HPLC peaks, and spurious results resulting from some artificial... 

Important. The substrate specificities and the product specificities of these enzymes determine the composition of volatile aldehydes formed from llnolelc acid and llnolenlc acid. The present report describes occurrence of, and substrate and product specificities of, lipoxygenase and hydroperoxide lyase In leaves, particularly In tea leaves. 

Table 3 Substrate specificity of tea chloroplast hydroperoxide lypse... 

The rate of hydroperoxide consumption, corresponding to lyase and dehydrase activities, was determined in different parts of the grain during germination (Fig. 1). Hydroperoxide lyase activity was measured in different parts of the grain but no activity was detected using the specific spectrophotometric assay. This indicates that hydroperoxide lyase is absent or exhibits low activity in the extracts (competition for the hydroperoxide substrate between hydroperoxide lyase and dehydrase may occur in the incubation media). Consequently, the consumption of hydroperoxides corresponds mainly to the dehydrase activity. 

Connell and Smithies (C2) have proposed a photometric peroxidase method with guaiacol as substrate and H202 instead of ethyl hydroperoxide. According to Nyman (N7), the method is less specific than Jayle s method. In the clinically interesting low range of HbBC (0-30 mg/100 ml) the method is unreliable. A simplification has been presented by Owen et al. (02). No report exists on its specificity, accuracy, or precision. 

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