Benzylhydrazine as a pseudo-substrate of bovine serum amine oxidase

Abstract

Bovine serum amine oxidase is inhibited by benzylhydrazine (BHy), but recovers full activity after a few hours incubation [Hucko-Haas & Reed (1970) Biochem. Biophys. Res. Commun. 38, 396-400]. The first phase of the process, requiring about 15 min, was found to consist of a mechanism-based hydrazine-transfer reaction leading to formation of the hydrazine-bound enzyme, benzaldehyde and H2O2. At variance with the enzymic process, the reaction with O2 preceded the benzaldehyde release. Two reaction intermediates could be characterized by optical spectroscopy and were assigned as the azo derivative and the benzaldehyde hydrazone, the latter one probably being involved in the reaction with O2. No reduction of Cu was detected at any stage. The hydrazine adduct could also be obtained by stoichiometric reaction of hydrazine with the native enzyme. The decay of this species occurred in about 8 h and was not studied in detail. The Cu-binding inhibitor NN-diethyldithiocarbamate affected the BHy reaction by stabilizing the benzaldehyde hydrazone form as against the azo derivative and the reaction with O2. However, under these same conditions the initial spectroscopic properties of the diethyldithiocarbamate adduct were recovered if the oxidase was left overnight. The reaction with O2 was abolished only upon removal of at least one Cu atom from the enzyme. On the basis of the failure to detect any change of Cu redox state and the enzyme behaviour in the presence of inhibitors, a reaction mechanism involving the formation of a hydroperoxy intermediate, as in the FAD-containing enzymes, is tentatively proposed.

The Reductive and Oxidative Half‐Reactions and the Role of Copper Ions in Plant and Mammalian Copper−Amine Oxidases

“…[51,54] According to Dooley, [54] the low concentration does not preclude a catalytic relevance of this radical, while Su and Klinman [51] proposed that the predominant Cu II Ϫaminoresorcinol is the species that reacts with O 2 forming a Cu II Ϫperoxide intermediate. The latter hypothesis is consistent with much experimental data that show Co IIsubstituted BSAO to be catalytically competent and thus suggest a metal structural function [42,66,69] and/or a Lewis acid role. [45] The nature of the radical in BSAO has recently been questioned, as it also forms when the enzyme is largely inactivated by reaction with hydrogen peroxide.…”

The oxidation and reduction reactions of bovine serum amine oxidase

“…This band is scarcely evident in our experiments because of the lower enzyme concentration employed; a similar problem has been reported by Olsson et al [13]. To obtain more information on the initial phases of the reaction of the amine substrate with BSAO, we have chosen the aromatic hydrazines as reasonable models of benzylamine, since their reaction with TPQ is irreversible over the time scale of stopped-flow experiments [15,16,18].…”

“…[13]. To obtain more information on the initial phases of the reaction of the amine substrate with BSAO, we have chosen the aromatic hydrazines as reasonable models of benzylamine, since their reaction with TPQ is irreversible over the time scale of stopped‐flow experiments [15,16,18].…”

Purification and Characterization of Pea Seedling Amine Oxidase for Crystallization Studies

“…This number agrees with the size determined by the translated gene sequence and carbohydrate content (M. McPherson, personal communication). Indeed, the recently published translated amino acid sequences of several other amine oxidases indicate subunit sizes in the 67-to 77-kD range (Bruinenberg et al, 1989;Rossi et al, 1992;Zhang et al, 1993), which are significantly smaller than those deduced by the more traditional techniques of gel filtration and Bhyd has been reported to be a slow substrate for bovine plasma amine oxidase (Morpurgo et al, 1989), yet it reacts irreversibly and stoichiometrically with the amine oxidase isolated from lentil seedlings (Padiglia et al, 1992). With PSAO, the Bhyd reaction was also irreversible and stoichiometric.…”