Peroxidase-like activity of lipoxygenases: different substrate specificity of potato 5-lipoxygenase and soybean 15-lipoxygenase and particular affinity of vitamin E derivatives for the 5-lipoxygenase.

Potato 5-lipoxygenase (5-PLO) catalyzes the reduction of 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid (13-HPOD) in the presence of vitamin E. I mol of vitamin E is required to consume 2 mol of 13-HPOD. The mechanism of the 5-PLO-catalyzed oxidation of vitamin E by 13-HPOD is similar to that previously established for the soybean 15-lipoxygenase (L-1)-catalyzed oxidation of phenidone by 13-HPOD, and seems to involve a one-electron reduction of the O-O bond of 13-HPOD. 5-PLO and L-1 exhibit very different substrate specificities and pH profiles for their peroxidase-like activity. Actually, among the 20 compounds containing various reducible functions and the 10 derivatives of vitamin E which have been studied, only four products containing hydrophobic long chains, ascorbic acid 6-palmitate, the trolox esters of octanol and undecanol, and vitamin E exhibit high peroxidase-like activities for 5-PLO. On the contrary, much more compounds, even not very hydrophobic, are good substrates for the peroxidase-like activity of L-1.

Effect of HIV-1 peptide presentation on the affinity constants of two monoclonal antibodies determined by BIAcore technology.

We studied two monoclonal antibodies (MAbs 9-11 and 41-1) which are specific for dominant and conserved epitopes located on HIV-1 transmembrane Gp41. These MAbs recognize both Gp41 and a synthetic HIV-1 envelope peptide (39GC) which is a fragment of Gp41. The interactions between MAbs 9-11 and 41-1 and 39GC either coupled to a sensor chip or to alkaline phosphatase were investigated using BIAcore technology. The association and dissociation rate constants as well as the affinity constants were determined. BIAcore technology allows real-time determination of the interaction between two molecules without the need for any labeling, neither isotopic nor enzymatic. The peptide 39GC was immobilized by coupling to dextran on the BIAcore biosensor through a disulfide bond with a cysteine residue added to the N-terminus of the synthetic peptide. The two native cysteine residues located in the loop of Gp41 were protected by ethylcarbamoyl residues (CONHC2H5); this chemical modification prevented the formation of the S-S bridge and in particular the internal loop. We specifically studied the interaction between the MAbs and either the protected peptide or the peptide whose cysteine residues had been deprotected in situ by alkaline treatment. The results showed that MAb 41-1 recognized 39GC either protected (Ka = 7.6 x 10(6) M-1) or unprotected (Ka = 1.48 x 10(8) M-1), whereas MAb 9-11 recognized only the unprotected form (Ka = 2.18 x 10(8) M-1). Our results suggest that the epitope MAb 9-11 is directed against a part of the peptide sequence which includes the two native cysteines. The difference in affinity observed for MAb 41-1 between the protected and the unprotected forms of 39GC was found to be due to a lower rate of dissociation for unprotected 39GC; these results illustrate the importance of peptide conformation on antibody recognition and might be explained by a conformational change due to reconstitution of the internal loop following deprotection of the thiol groups. MAbs 9-11 and 41-1 also recognized 39GC conjugated to alkaline phosphatase and deprotected. We observed a difference between the rate constants for MAb 41-1 binding to free peptide and its binding to the peptide-enzyme conjugate which might be due to changes in peptide flexibility. In contrast, the rate constants of MAb 9-11 were the same in both experiments, suggesting that the rigidity of the internal loop prevents changes in 9-11 epitope conformation.

Porcine leukocyte 5- and 12-lipoxygenases are iron enzymes.

5- and 12-lipoxygenases isolated from porcine leukocytes were investigated by electron paramagnetic resonance at X-band and atomic absorption spectroscopy. For comparison potato 5-lipoxygenase was studied under identical experimental conditions. All three lipoxygenases contained between 0.7 and 0.9 Fe atoms/enzyme molecule. As isolated, both mammalian enzymes exhibited a characteristic EPR signal at low magnetic field with a maximum at g = 5.20 indicative of a high-spin ferric iron center. The signal was not affected by the oxidants 12-hydroperoxyeicosatetraenoic acid or arachidonic acid, nor was it affected by the reductant nordihydroguaiaretic acid. In the case of the potato enzyme an intense EPR signal with resonances at g = 7.50, 6.39 and 5.84 was only observed after addition of an oxidant, such as 9-hydroperoxyoctadecadienoic acid.

Soybean lipoxygenase-catalyzed oxidations by linoleic acid hydroperoxide: different reducing substrates and dehydrogenation of phenidone and BW 755C.

Phenidone is not a substrate for dioxygenation by soybean lipoxygenase-1 (L1) but reduces L1Fe(III) into L1Fe(II), as shown by EPR spectroscopy. L1 catalyzes the oxidation of phenidone by 13-HPOD, the hydroperoxide formed by dioxygenation of linoleic acid by L1, with formation of 4,5-dehydrophenidone. Two moles of 13-HPOD are used per mole of phenidone dehydrogenated. Other pyrazoline derivatives such as BW 755C, but also, in a more general manner, different compounds containing phenol, aniline, hydrazine, hydroxylamine or hydrazide functions act as reducing substrates for decomposition of 13-HPOD by L1.

Mechanisms of inactivation of lipoxygenases by phenidone and BW755C.

Inhibition of soybean lipoxygenase (L-1) and potato 5-lipoxygenase (5-PLO) by the pyrazoline derivatives phenidone and BW755C only occurs after oxidation of these compounds by the peroxidase-like activity of the lipoxygenases. There is a clear relationship between this oxidation and the irreversible inactivation of L-1. The final product of phenidone oxidation by L-1, 4,5-didehydrophenidone, is not responsible of this inactivation, but the species derived from a one-electron oxidation of phenidone plays a key role in L-1 inactivation. In the absence of O2, inactivation of 1 mol of L-1 occurs after the oxidation of 34 mol of phenidone and the covalent binding of 0.8 mol of phenidone-derived metabolite(s) to L-1. In the presence of O2, inactivation of 1 mol of L-1 occurs already after oxidation of 11 mol of phenidone and only involves the covalent binding of 0.4 mol of phenidone-derived metabolite(s) to L-1. A mechanism is proposed for L-1 inactivation by phenidone, which involves the irreversible binding of a phenidone metabolite to the protein and the oxidation of an L-1 amino acid residue (in the presence of O2).

Soybean lipoxygenases-1, -2a, -2b and -2c do not contain PQQ.

Soybean isoenzymes lipoxygenases-1, -2a, -2b and -2c were examined spectroscopically for the presence of covalently bound pyrrolo quinoline quinone (PQQ) after derivatization by phenylhydrazine (PH), 2,4-dinitrophenylhydrazine (DNPH) and 3-methyl-2-benzothiazolinone hydrazone (MBTH). DNPH derivatization of PQQ after a pronase digestion step of lipoxygenase-1 in the presence of an anion exchange gel fixing the cofactor was also investigated. None of these experiments provided evidence for the presence of PQQ contrary to previous report by Van der Meer et al (1). We have checked, by EPR spectroscopy, that the three reactants used were able to reduce the active site ferric iron. Our results were confirmed by the absence of enzyme inhibition by cis- and trans-1,2-diaminocyclohexane or benzylamine in the presence of NaBH3CN which have been reported to react with PQQ and to inactivate quinoproteins (2,3).