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Aromatic hydrazides of the general formula NH2NHCO(CH2)nC6H4R were covalently bound by bovine serum amine oxidase (BSAO), giving rise to optical and CD absorptions at 350-400 nm. Benzohydrazides (n = 0) reacted slowly, in the ratio of one per dimeric protein molecule, like semicarbazide. Phenylacetohydrazides (n = 1) and phenylpropionic hydrazides (n = 2) reacted instead in the ratio of two per dimer, one molecule at a much faster rate than the other. The fast reaction correlated with the loss of enzymatic activity. The contribution to the optical absorbance of either molecule was identical, but only the first one produced a CD band, the wavelength and sign of which were determined by the number n of methylene groups in the hydrazide. In n = 1 and n = 2 compounds, the reaction was faster as the R substituent became more hydrophobic (triazolyl less than imidazolyl less than phenyl), suggesting a specific interaction with the protein matrix. Phenylhydrazine was found to react with the native enzyme in the ratio of only one per protein dimer. However, one phenylhydrazine was also slowly bound by most 1:1 enzyme-hydrazide adducts, with the formation of ternary derivatives. Phenylhydrazine formed the usual intense band at 447 nm with n = 1 and n = 2 hydrazide-BSAO adducts and a weaker, blue-shifted band with the adducts of semicarbazide and of some n = 0 hydrazides. In both cases, the hydrazide absorption band was unaffected. Competition was observed with other benzohydrazides and with the second molecule of n = 1 compounds. A half-site mechanism appears to be operative, the second site being always less reactive than the first. Reactivity and adduct conformation were also affected by N,N-diethyldithiocarbamate, a powerful enzyme inhibitor that binds copper.

The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence of N,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.

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.

A method is proposed for the determination of the activity of amine oxidases in purified samples and tissue homogenates. The method is based on the chemiluminescence of luminol and other cyclic hydrazides elicited by the horseradish peroxidase-catalyzed peroxidation using H2O2 produced in the amine oxidase reaction. Several aspects of the chemiluminescence method for determining enzymatic activity in crude tissue extracts are discussed.

The carbonyl cofactor of bovine serum amine oxidase, recently identified as pyrroloquinoline quinone [Ameyama, Hayashi, Matsushita, Shinagawa & Adachi (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, Jongejan, Frank & Duine (1984) FEBS Lett. 170, 305-309], reacts stoichiometrically and irreversibly with hydrazides of phenylacetic acid and of benzoic acid. With the phenylacetic hydrazides a reversible intermediate step was detected by competition with substrate, carbonylic reagents or phenylhydrazine, a typical inhibitor of the enzyme. All hydrazides form an intense broad band with maximum absorbance in a narrow wavelength range (350-360 nm), irrespective of the acyl group, suggesting that the transition is located on the organic cofactor. A different situation is found with some phenylhydrazines, where extended conjugation can occur between the cofactor and the phenyl pi-electron system via the azo group, as shown by the lower energy and higher intensity of the transition. In this case the transition is sensitive to substituents in the phenyl ring. The c.d. spectrum of the adducts is influenced by the type of hydrazide (derived from phenylacetic acid or benzoic acid), by pH and by NN-diethyldithiocarbamate binding to copper, probably as a result of shifts of equilibria between hydrazone-azo tautomers.

An oxygraphic method for the assay of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase activity which makes use of the coupled reactions of Dopa decarboxylase with serum amine oxidase is presented. Both free and immobilized amine oxidases were utilized. The assay is simple, rapid, and allows a continuous monitoring of the reaction. The kinetic parameters for Dopa decarboxylase obtained with the coupled assay do not significantly differ from those obtained by standard methods.

Some hydrazides of pyrrol-1-ylbenzoic and pyrrol-1-ylphenylacetic acids were prepared, and their effect on copper-dependent amine oxidases (Cu-AOs) and FAD monoamine oxidases (MAOs) activities was tested. The compounds were not substrates for Cu-AO enzymes but acted as noncompetitive inhibitors. Hydrazides of pyrrol-1-ylphenylacetic acids were highly specific for plasma amine oxidase (Ki = 0.5-1 microM). In contrast, all the hydrazides were weak inhibitors of MAO activity. Incubation with the hydrazide derivatives led to irreversible inactivation of Cu-AOs. Therefore, the inhibition implied two distinct steps. The first one consisted of the rapid formation of the enzyme-inhibitor complex and was reversed by dialysis. In the second step, the complex was irreversibly transformed, probably by the formation of a Schiff base between the hydrazide and the prosthetic carbonyl group of the enzyme.