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As yet, the physiological significance of hydroxylation of anandamide and linoleoyl amides is unknown. Therefore, we investigated whether hydroxylation of ODNHEtOH and ODNH2 influences their binding abilities to the CB-1 receptor and whether it alters their reactivity towards a fatty acid amide hydrolase (FAAH) from rat brain. Neither the fatty acid amides nor their hydroxylated derivatives were able to displace the potent cannabinoid [3H]CP 55.940 from the CB-1 receptor (Ki > 1 microM). Hydroxylation of ODNHEtOH resulted in a strong reduction of the maximum rate of hydrolysis by a FAAH, but the affinity of FAAH for the substrate remained of the same order of magnitude. Hydroxylation of ODNH2 led to a decrease in the affinity of FAAH for the substrate, but its maximum rate of conversion was unaffected. Furthermore, hydroxylation of ODNHEtOH enhanced its capacity to inhibit competitively the hydrolysis of anandamide. The resulting prolonged lifetime of anandamide and other fatty acid amide derivatives may have a considerable impact on cellular signal transduction.

Anandamide, a novel neurotransmitter, has been reported to be dioxygenated by brain lipoxygenase [1,11]. Anandamides constitute a new class of neuroregulatory fatty acid amides. However, little is known about the enzymatic dioxygenation of these lipids. Therefore, we have tested several members of the neuroactive fatty acid amide class containing a 1Z,4Z-pentadiene system whether they could be dioxygenated by soybean lipoxygenase-1, which is a model enzyme for mammalian lipoxygenases. In this study it was found that lipoxygenase-1 converts N-linoleoylethanolamide (ODNHEtOH), N-linoleoylamide (ODNH2), N-linoleoylmethylamide (ODNHMe) and N,N-linoleoyldimethylamide (ODN(Me)2 into 13-(S)-hydroperoxy-9Z,11E-octadeca-9,11-dienoyl amides derivatives. The apparent Km values for ODNHEtOH (23.6 +/- 3.7 microM), ODNH2 (8.60 +/- 0.65 microM) and linioleic acid (OD: 8.85 +/- 0.74 microM) are not significantly different. The k(cat) for ODNH2 (32.4 +/- 1.2 s(-1)) is twice as small as compared to the turnover numbers of the other substrates, viz. ODNHEtOH (61.6 +/- 5.0 s(-1)) and OD (54.4 +/- 2.0 s(-1). The results suggest that N-linoleoyl ethanolamide and N-linoleoyl amide can be readily converted by lipoxygenases in vivo.

Triple bond analogues of natural fatty acids irreversibly inactivate lipoxygenase during their enzymatic conversion [Nieuwenhuizen, W. F., et al. (1995) Biochemistry 34, 10538-10545]. To gain insight into the mechanism of the irreversible inactivation of soybean lipoxygenase-1, we studied the enzymatic conversion of two linoleic acid analogues, 9(Z)-octadec-9-en-12-ynoic acid (9-ODEYA) and 12(Z)-octadec-12-en-9-ynoic acid (12-ODEYA). During the inactivation process, Fe(III)-lipoxygenase converts 9-ODEYA into three products, i.e. 11-oxooctadec-9-en-12-ynoic acid, racemic 9-hydroxy-10(E)-octadec-10-en-12-ynoic acid, and racemic 9-hydroperoxy-10(E)-octadec-10-en-12-ynoic acid. Fe(II)-lipoxygenase does not convert the inhibitor and is not inactivated by 9-ODEYA. Fe(III)-lipoxygenase converts 12-ODEYA into 13-hydroperoxy-11(Z)-octadec-11-en-9-ynoic acid (34/66 R/S), 13-hydroperoxy11(E)-octadec-11-en-9-ynoic acid (36/64 R/S), 11-hydroperoxyoctadec-12-en-9-ynoic acid (11-HP-12-ODEYA, enantiomeric composition of 33/67), and 11-oxooctadec-12-en-9-ynoic acid (11-oxo-12-ODEYA) during the inactivation process. Also, Fe(II)-lipoxygenase is inactivated by 12-ODEYA. It converts the inhibitor into the same products as Fe(III)-lipoxygenase does, but two additional products are formed, viz. 13-oxo-11(E)-octadec-11-en-9-ynoic acid and 13-oxo-11(Z)-octadec-11-en-9-ynoic acid. The purified reaction products were tested for their lipoxygenase inhibitory activities. The oxo compounds, formed in the reaction of 9-ODEYA and 12-ODEYA, do not inhibit Fe(II)- or Fe(III)-lipoxygenase. The 9- and 13-hydroperoxide products that are formed from 9-ODEYA and 12-ODEYA, respectively, oxidize Fe(II)-lipoxygenase to its Fe(III) state and are weak lipoxygenase inhibitors. 11-HP-12-ODEYA is, however, the most powerful inhibitor and is able to oxidize Fe(II)-lipoxygenase to Fe(III)-lipoxygenase. 11-HP-12-ODEYA is converted into 11-oxo-12-ODEYA by Fe(III)-lipoxygenase. We propose a mechanism for the latter reaction in which Fe(III)-lipoxygenase abstracts the bisallylic hydrogen H-11 from 11-HP-12-ODEYA, yielding a hydroperoxyl radical which is subsequently cleaved into 11-oxo-ODEYA and a hydroxyl radical which may inactivate the enzyme.

Fe K-edge X-ray absorption spectra of the non-heme iron constituent of lipoxygenase-1 from soybeans were obtained. The spectrum of 2.5 mM Fe(II) lipoxygenase, mixed with 1.2 M linoleate in the absence of O2, was compared to the spectrum of the native (i.e. untreated) enzyme. In the lipoxygenase-linoleate complex, an edge shift to lower energy was observed. This indicated that the iron-ligand distances in this complex are slightly longer than those in the untreated enzyme species. The extended X-ray absorption fine structure spectrum of Fe(II) lipoxygenase, prepared by anaerobic reduction of 2.5 mM Fe(II) lipoxygenase with 1.2 M linoleate, was very similar to the spectrum of the anaerobic lipoxygenase-linoleate complex. We conclude that the conformational differences between the iron coordination spheres of native and cycled Fe(II) lipoxygenase must be ascribed to the presence of linoleate, and not to changes in the enzyme that occur only after one cycle of oxidation and reduction. Furthermore, spectra of 2.5 mM Fe(II) lipoxygenase mixed with 1.2 M oleate, either in the absence or in the presence of O2, were also identical to the spectrum of the Fe(II) lipoxygenase-linoleate complex. This finding is in agreement with our observation that oleate is a competitive inhibitor of the lipoxygenase reaction. Moreover, the similarity of the lipoxygenase-oleate complexes in the presence and absence of O2 excludes the possibility that O2 binding to the iron cofactor is induced upon binding of a fatty acid to lipoxygenase.

In order to investigate the activation of lipoxygenase and to clarify the role of the oxygenation product hydroperoxide in this process, the effect of 13-hydroperoxylinoleic acid (P, 0-35 microM) on linoleic acid (S, 1-80 microM) oxygenation catalysis by 12 nM lipoxygenase-1 from soybean was studied at pH 10, 25 degrees C, and 240 microM O2 with rapid kinetic techniques. The following observations were made: (1) Iron(II) and iron(III) lipoxygenases are kinetically different: reactions started with the Fe(II) enzyme form show a lag phase, whereas iron(III) lipoxygenase induces an initial burst. (2) Oxidation of the enzyme alone is not sufficient to abolish the lag phase: at [S] greater than 50 microM, the initial burst in the iron(III) lipoxygenase curves is still followed by a lag. The lag phase disappears completely only in the presence of micromolar quantities of P. (3) The approximate dissociation constants for S and P are 15 and 24 microM, respectively, 1 order of magnitude smaller than the corresponding values in the absence of oxygen. The observed kinetics are predicted by numerical integration of the rate equations of a model based on the single lipid binding site mechanism for the anaerobic lipoxygenase reaction [Ludwig et al. (1987) Eur. J. Biochem. 168, 325-337; Verhagen et al. (1978) Biochim. Biophys. Acta 529, 369-379]. A quasi-steady-state approximation of the model suggests that a high [S]/[P] the fraction of active iron(III) lipoxygenase is small and that, therefore, a lag phase is intrinsic to the mechanism.

1. The addition of 13-Ls-hydroperoxylinoleic acid to lipoxygenase-1 (linoleate: oxygen oxidoreductase EC 1.13.11.12) from soybeans at pH 9 and 25 degrees C causes a quenching of the fluorescence of the enzyme at 328 nm when exciited at 280 nm and gives rise to an increase of the absorbance of the enzyme in the 300 nm to 450 nm region. 2. In the absence of 02, addition of linoleic acid to enzyme treated with 13-Ls-hydroperoxylinoleic acid, causes an increase of the fluorescence at 328 nm and a decrease of the absorbance in the 300 nm to 450 nm region. 3. The fluorescence changes are suggested to be directly coupled to the absorbance changes via a non-radioactive energy transfer process. 4. It is proposed that the observed fluorescence and absorbance changes are related to changes in the formal change of iron in the protein.

Lipoxygenase-1 from soybeans is incubated with an isomer of linoleic acid, 13-cis, 16-cis-octadecadienoic acid. Analysis of the oxygenation products indicates that molecular oxygen is stereospecifically introduced mainly at C-17 (n-2) of the fatty acid (in the LS-configuration), and only to a minor extent at C-13 (n-6). These findings contradict previous suggestions about the postional specificity of lipoxygenase-1.

The steady-state kinetics of the anaerobic reaction of soybean lipoxygenase-1 with linoleic acid and 13-L-hydroperoxylinoleic acid were studied. Initial rates of the formation of oxodienoic acids**, absorbing at 285 nm, were measured at pH 10. About 50% of the consumed 13-L-hydroperoxylinoleic acid was converted into oxodienoic acids regardless of the initial ratio of the two substrates. A linear inhibition by both linoleic acid and 13-L-hydroperoxylinoleic acid was observed in the concentration range studied, which is on the upper side limited by the concentrations at which micelle- or acid-soap formation starts. A kinetic scheme is proposed based on one active site in lipoxygenase-1 which alternately binds the two substrates. Values for the kinetic constants were calculated by fitting simultaneously the complete set of data to the appropriate rate equation.

1. The mode of action of flax-seed hydroperoxide isomerase was studied in vitro by using as substrates linoleic acid hydroperoxides formed by soya-bean lipoxygenase. 2. The enzyme converts only 13-hydroperoxyoctadeca-cis-9-trans-11-dienoic acid, whereas the 9-hydroperoxy isomer does not react. 3. The isomerization product was identified by chemical and spectroscopic methods as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid. 4. 12,13-Epoxyoleic acid isomers do not act as intermediates in the isomerization reaction. 5. Suggestions for a functional relationship between hydroperoxide isomerase and lipoxygenase are discussed.