Hexanal phenylhydrazone is a mechanism-based inactivator of soybean lipoxygenase 1.
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.
Galey
J B
JB
Laboratoire de chimie et biochimie pharmacologiques et toxicologiques, Unité associée au CNRS en développement concerté avec l'INSERM (UA 400), Université René Descartes, Paris, France.
Bombard
S
S
Chopard
C
C
Girerd
J J
JJ
Lederer
F
F
Thang
D C
DC
Nam
N H
NH
Mansuy
D
D
Chottard
J C
JC
D004578:Electron Spin Resonance Spectroscopy; D005979:Glutathione Peroxidase / Q000378:metabolism; D006835:Hydrazones / Q000138:chemical synthesis / Q000494:pharmacology*; D007700:Kinetics; D008084:Lipoxygenase / Q000302:isolation & purification; D016859:Lipoxygenase Inhibitors; D010944:Plants / Q000201:enzymology*; D013025:Glycine max
D006835:Hydrazones; D016859:Lipoxygenase Inhibitors; C055644:hexanal phenylhydrazone; D005979:Glutathione Peroxidase; D008084:Lipoxygenase