Spectroscopic studies of pig kidney diamine oxidase-anion complexes

Complexes of pig kidney diamine oxidase with azide, thiocyanate, and cyanide have been characterized by EPR and circular dichroism spectroscopy. Cu(II) d-d bands are observed in the CD spectrum of the resting enzyme at ≈800 nm (12 500 cm⁻¹) and 575 nm (17 400 cm⁻¹). Anion binding produces characteristic changes in the CD spectra. N₃⁻/SCN⁻ → Cu(II) ligand-to-metal charge-transfer transitions are located at 390 nm (25 600 cm⁻¹) and 365 nm (27 400 cm⁻¹), respectively. In addition, an intense new band grew in at ≈500 nm (20 000 cm⁻¹) when azide or thiocyanate were added, which may be assigned as a Cu(II) electronic transition that gains rotational strength in the anion complex. EPR spectra established that the Cu(II)-anion complexes are tetragonal; however, the magnitudes of the anion-induced shifts in the EPR parameters were consistent with substantial perturbations of the Cu(II) electronic ground state in the thiocyanate and cyanide complexes. Prominent superhyperfine splitting was apparent in the EPR spectra of the diamine oxidase complexes with thiocyanate and cyanide. The superhyperfine structure is (at least) partially attributable to endogenous Cu(II) ligands, probably from imidazole.     

Oxidation of some seleno-diamines by pig kidney diamine oxidase

The enzymatic deamination of Se-lanthionamine (Se-L), Se-homolanthionamine (Se-HL) and Se-homocystamine (Se-HC) catalyzed by pig kidney diamine oxidase (PKAO) has been studied to collect information about the possible metabolic reactions of selenocompounds in comparison with the sulfur ones. The results indicate that: Se-L, Se-HL and Se-HC are substrates for PKAO. The first product of Se-L, Se-HL and Se-HC oxidative deamination, in analogy with other thio and seleno diamines, may be the cyclized aminoaldehyde. The final product of Se-L, Se-HL and Se-HC degradation are ammonia, elemental selenium and C2 or C3 compounds.     

Inhibition of pig kidney diamine oxidase by substrate analogues

1. The oxidation of p-dimethylaminomethylbenzylamine was followed spectrophotometrically by measuring the change in E(250) caused by the p-dimethylaminomethylbenzaldehyde produced. 2. This reaction was inhibited by substrate analogues such as isothiouronium, guanidino, dimethylsulphonium and trimethylammonium compounds. 3. The inhibition by both mono- and bis-onium compounds has been studied and a comprehensive theory is developed to explain both the type and degree of inhibition produced.     

Kinetic studies on the inhibition mechanism of diamine oxidase from porcine kidney by aminoguanidine

The mechanism of inhibition of diamine oxidase [DAO, histaminase, amine: oxygen oxidoreductase (deaminating) (copper-containing), EC 1.4.3.6] by aminoguanidine was studied kinetically. The DAO reaction was carried out with putrescine as a substrate in the presence of o-aminobenzaldehyde and followed by the continuous increase in A430. An enzymatic reaction product, 4-aminobutyraldehyde, was spontaneously cyclized to delta 1-pyrroline, which coupled with o-aminobenzaldehyde to give rise to a yellow quinazolinium chromophore [Holmstedt et al. (1961) Biochim. Biophys. Acta 48, 182-186]. First, to measure the initial velocities as accurately as possible, the kinetic relationship between the oxidase reaction rate and the color development rate was formulated. The reaction proceeded linearly in the absence of aminoguanidine, but slowed down time-dependently in its presence. When the color development velocities in the presence of the inhibitor were plotted against time, the plot was nonlinear and there seemed to be at least two phases of the inhibition. Nonlinear least-squares analysis of the data dA430/dt showed that the simplest model that fitted best was a model composed of two species of enzyme-inhibitor complexes: E + I k+1 in equilibrium k-1 EI k+2 in equilibrium k-2 E'I where E is the enzyme, I is aminoguanidine (an inhibitor), and the ks are the rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and oxidation of aminoalkyl-onium compounds by pig kidney diamine oxidase

1. The preparation of a series of compounds derived from diamines by replacing one amino group by a dimethylsulphonium, isothiuronium, trimethylammonium, NN'-dimethylimidazolium or N-methylpyridinium species is described. 2. The behaviour of these compounds as substrates of pig kidney diamine oxidase is reported. All but the trimethylammonium compounds proved to be substrates. 3. Many of these compounds showed potent inhibition at high substrate concentration and this was studied. 4. On the basis of these and other observations a scheme for enzyme-substrate interaction is suggested.     

Salt effect on pig kidney diamine oxidase activity

The aim of the present investigation was to study specific effects of ionic strength on the amine oxidase activity. Different patterns were shown in the variation of enzymatic activity, according to the cationic species and the substrates tested (cadaverine and butylamine). Different cations exert specific effects, particularly sodium, which seems to regulate in some way the diamine oxidase activity (Fig. 1 and 2). Involvement of active sites of enzyme interacting with charged molecules is postulated.     

Analytical-band centrifugation of the active form of pig kidney diamine oxidase

In this communication it is shown that pig kidney diamine oxidase undergoes an association-dissociation reaction which is under the influence of the concentration of oxygen, one of the substrates. The sedimentation constant of the active unit was measured using the analytical-band centrifugation of the active enzyme-substrate complex.     

A reinvestigation of the substrate specificity of pig kidney diamine oxidase

1. The substrate specificity of pig kidney diamine oxidase was reinvestigated with a purer enzyme preparation than has previously been used for this purpose. 2. All substrates were extensively purified before use, and methods of preparation or sources are given, together with R(F) values. 3. The substrate specificity determined differed somewhat from that reported by previous workers and, in addition, the behaviour of several compounds not previously used as substrates is described. 4. A model for enzyme-substrate interaction embodying these observations is formulated. It is suggested that a negatively charged substrate-binding group is situated at 6.0-9.0 A from the oxidizing site. The binding and oxidizing sites are separated by a hydrophobic or methylene-binding site.     

On the nature of chromophore in pig kidney diamine oxidase.

The nature of the 500-nm chromophore in pig kidney diamine oxidase was investigated by absorption spectroscopy and fluorescence in the presence of various chelating or carbonyl-specific reagents. From the spectroscopic measurements the following conclusions can be drawn. First, the 500-nm absorption band is not due to copper, the reduction of which is not related to the disappearance of this band. Second, phenylhydrazine and cycloserine give rise, upon reaction with the enzyme, to absorptions very similar to those of a pyridoxal enzyme, aspartate aminotransferase. Third, these enzyme derivatives are unexpectedly non-fluorescent. Copper removal, obtained after prolonged incubation of cycloserine-treated enzyme in the presence of reducing and chelating agents, leads to a fluorescence similar to that of cycloserine-aspartate transminase. It is proposed that copper is coordinated to the postulated pyridoxal phosphate of diamine oxidase through the pyridine nitrogen.     

Immunochemical reactivity of native and modified preparations of pig kidney diamine oxidase

This paper describes the antigenicity of pig kidney diamine oxidase [EC 1.4.3.6] and the possible role of constituent amino acids in the epitope structure of the enzyme. The loss of 62% of the biological activity after DAO-anti-DAO antibodies interaction was attributed to the steric hindrance caused by binding of antibody to the enzyme molecule. A gradual loss in antigenicity during ultraviolet (UV) irradiation was observed without any significant conformational change, demonstrating the destruction of antigenic determinants. However, ethoxyformylation of nine histidyl residues with complete inactivation caused no change in immunoreactivity. The results indicate that the antigenic sites and catalytic sites are located at different positions along the polypeptide chain. Moreover, the results of lysine residue modification were suggestive of possible involvement of lysine in the antigenic determinants of DAO.     

Oxidation of p-dimethylaminomethylbenzylamine by pig kidney diamine oxidase. A new method for spectrophotometric assay

1. The oxidation of some para-substituted benzylamines by diamine oxidase produces the corresponding aldehydes. This was studied to develop a spectrophotometric method for following the enzyme reaction, as the aldehydes produced absorb strongly at 250nm where the substrates are almost optically transparent. 2. p-Dimethylaminomethylbenzylamine was the most useful substrate and full details of its preparation are given. The synthesis of its related oxidation product, p-dimethylaminomethylbenzaldehyde, is also described. 3. The effects of variations in pH, ionic strength, temperature and oxygenation on the reaction are described and the usefulness of the method is illustrated by several applications and assessed by comparison with the standard spectrophotometric assay.     

Equations of substrate-inhibition kinetics applied to pig kidney diamine oxidase (DAO, E.C. 1.4.3.6)

Pig kidney diamine oxidase (DAO) and other semicarbazide-sensitive amine oxidases (SSAO) show clear substrate-inhibition kinetics and a reaction-scheme mechanism based on two substrate binding sites. We evaluated several reaction scheme mechanisms with a non-linear regression program (NCSS), estimating R2, the constants of the equations and their standard errors and we determined the deviation of experimental data from theoretical equations. The best fit was obtained with a "dead end" mechanism with two binding sites. Based on this scheme, other schemes for a two-substrate reaction and for mechanisms of inhibition were constructed. These reaction schemes, even at low substrate concentration, fitted experimental data better than Michaelis-Menten kinetics, and provided information on the mechanisms of action of inhibitors. The presence of two substrate-binding sites on pig kidney DAO was confirmed by all experimental data.