Hexanal phenylhydrazone is a mechanism-based inactivator of soybean lipoxygenase 1

Abstract

Hexanal phenylhydrazone (1; 70:30 E:Z mixture) at micromolar concentration irreversibly inactivates soybean lipoxygenase 1 (L-1) in the presence of dioxygen. L-1 catalyzes the oxidation of 1 into its alpha-azo hydroperoxide 2 [C5H11CH(OOH)N = NC6H5]. 2 is an efficient inactivator of L-1. The aerobic reaction between 1 and L-1 follows a branched pathway leading to the release of 2 into the medium or to L-1 inactivation. The respective parameters corresponding to this inactivation by the (E)-1 and (Z)-1 isomers are Ki = 0.25 and 0.40 microM and kinact = 0.8 and 2.1 min-1. Linoleic acid protection agrees with a mechanism-based inactivation process. The oxidation of a minimum of 13 +/- 3 molar equiv of 1 is required for complete L-1 inactivation, but up to 70 equiv is necessary in the presence of a very large excess of 1. The inactivation is actually the result of two pathways: one is due to a reaction of 2 as soon as it is formed at the active site (20%); the other is due to 2 released into the medium and coming back to the active site (80%). The inactivation is accompanied by the oxidation of 1.8 +/- 0.8 methionine residues of the protein into the corresponding sulfoxide. The inactivated L-1 is electron paramagnetic resonance (EPR) silent with an effective magnetic moment of mu = 5.0 +/- 0.1 Bohr magnetons corresponding to an S = 2 spin state. An inactivation mechanism is proposed on the basis of EPR and magnetic susceptibility data obtained from the anaerobic and aerobic reactions of L-1 with 1 and 2.

Phenylhydrazones as new good substrates for the dioxygenase and peroxidase reactions of prostaglandin synthase: Formation of iron(III)-.sigma.-phenyl complexes

“…Possible products of dioxygenation of 1 by 0 2 catalyzed by PGHS were the corresponding a-azo hydroperoxide and benzaldehyde by analogy to what was previously found for the oxidation of phenylhydrazones by 0 2 catalyzed by other dioxygenases, the lipoxygenases (eq 2) (Galey et al, 1988). hydroperoxide 3 (PhCH(OOH)N=NPh) or of the corresponding a-azo alcohol 4 (PhCH(OH)N=NPh) also exhibit UV spectra characterized by a 277-nm peak (Yao & Resnick, 1965).…”

“…corresponding cy-azo hydroperoxides was found to be catalyzed by lipoxygenases (Galey et al, 1988). Its mechanism should be similar to that proposed for the dioxygenation of unsaturated fatty acids, except that the hydrogen abstracted by the PGHS active species is at a benzylic-allylic N H site instead of a bis-allylic CH2 site.…”

Prevalence of anal fistula in the United Kingdom

“…Reported population-based incidence estimates for anal fistula in CD patients range between 1.2%[37] to 7.5%[38]. Based on these values, a systematic review calculated anal fistula prevalence among CD patients to be 3.4%-6.0% in the Europe[8].…”

Lipoxygenase - a versatile biocatalyst for biotransformation of endobiotics and xenobiotics

“…Both the parent compound and its oxidation product exhibit a potent, selective inhibitory effect on the dioxygenase activity while the hydroperoxidase activity is spared [149]. Similar to ETI and ETYA, the oxidation of hexanal phenylhydrazone leads to the inactivation of SLO due to the concomitant oxidation of the methionine residue of the protein to corresponding sulfoxide [79]. SLO is rapidly inactivated when incubated with arachidonic acid and either NDGA, the aminopyrazolines BW 755C and BW 540C, or the acetohydroxamic acid derivatives BW A4C and BW A137C [150].…”