Copper-dependent Amine Oxidase Research Articles

Mechanisms of metalloenzymes studied by quantum chemical methods.

The study of metalloenzymes using quantum chemical methods of high accuracy is a relatively new field. During the past five years a quite good understanding has been reached concerning the methods and models to be used for these systems. For systems containing transition metals hybrid density functional methods have proven both accurate and computationally efficient. A background on these methods and the accuracy achieved in benchmark tests are given first in this review. The rest of the review describes examples of studies on different metalloenzymes. Most of these examples describe mechanisms where dioxygen is either formed, as in photosystem II, or cleaved as in many other enzymes like cytochrome c oxidase, ribonucleotide reductase, methane mono-oxygenase and tyrosinase. In the descriptions below high emphasis is put on the actual determination of the transition states, which are the key points determining the mechanisms.

Aminoacetone Induces Loss of Ferritin Ferroxidase and Iron Uptake Activities

Aminoacetone (AA) is a threonine and glycine metabolite overproduced and recently implicated as a contributing source of methylglyoxal (MG) in conditions of ketosis. Oxidation of AA to MG, NH4+, and H2O2 has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by copper- and iron ion-catalyzed reactions with oxygen. We previously demonstrated that AA-generated O2*-. and enoyl radical (AA*) induce dose-dependent Fe(II) release from horse spleen ferritin (HoSF); no reaction occurs under nitrogen. In the present study we further explored the mechanism of iron release and the effect of AA on the ferritin apoprotein. Iron chelators such as EDTA, ATP and citrate, and phosphate accelerated AA-promoted iron release from HoSF, which was faster in horse spleen isoferritins containing larger amounts of phosphate in the core. Incubation of apoferritin with AA (2.5-50 mM, after 6 h) changes the apoprotein electrophoretic behavior, suggesting a structural modification of the apoprotein by AA-generated ROS. Superoxide dismutase (SOD) was able to partially protect apoferritin from structural modification whereas catalase, ethanol, and mannitol were ineffective in protection. Incubation of apoferritin with AA (1-10 mM) produced a dose-dependent decrease in tryptophan fluorescence (13-30%, after 5 h), and a partial depletion of protein thiols (29% after 24 h). The AA promoted damage to apoferritin produced a 40% decrease in apoprotein ferroxidase activity and an 80% decrease in its iron uptake ability. The current findings of changes in ferritin and apoferritin may contribute to intracellular iron-induced oxidative stress during AA formation in ketosis and diabetes mellitus.

Somatic mutations of the lysyl oxidase gene on chromosome 5q23.1 in colorectal tumors.

Lysyl oxidase (LOX), a copper-dependent amine oxidase, has been implicated in tumor suppression and cell growth regulation. The chromosomal locus of LOX, 5q23, is affected by loss of heterozygosity (LOH) in colon cancer, suggesting that the LOX gene could be affected by LOH and consequently, loss or reduction of LOX function contribute to the tumorigenic process. Identification of microsatellite markers within the LOX locus has allowed us to map the LOX gene within the 5q23.1 region. Analysis of this locus and flanking loci in matched tumor and blood DNA samples from a panel of colorectal cancer patients, demonstrated that 38% (16/42) of informative samples were affected by LOH or allelic imbalance. Furthermore, 75% (6/8) of these tumor samples were shown to have significantly reduced LOX mRNA levels. Similar reduction in LOX levels were detected in a panel of matched normal colon and colon tumor samples. Tumor samples demonstrating LOH by RFLP, were subject to mutational analysis, including RT-PCR, exonic deletion detection by PCR, cDNA and genomic DNA sequencing, and were found to have a spectrum of alterations and mutations affecting the LOX gene. These results confirm that loss or reduction of LOX function during tumor development is a direct consequence of somatic mutations and is associated with colon tumor pathogenesis.

Aerobic oxidation of aminoacetone, a threonine catabolite: iron catalysis and coupled iron release from ferritin.

Aminoacetone (AA) is a threonine and glycine catabolite long known to accumulate in cri-du-chat and threoninemia syndromes and, more recently, implicated as a contributing source of methylglyoxal (MG) in diabetes mellitus. Oxidation of AA to MG, NH(4)(+), and H(2)O(2) has been reported to be catalyzed by a copper-dependent semicarbazide sensitive amine oxidase (SSAO) as well as by Cu(II) ions. We here study the mechanism of AA aerobic oxidation, in the presence and absence of iron ions, and coupled to iron release from ferritin. Aminoacetone (1-7 mM) autoxidizes in Chelex-treated phosphate buffer (pH 7.4) to yield stoichiometric amounts of MG and NH(4)(+). Superoxide radical was shown to propagate this reaction as indicated by strong inhibition of oxygen uptake by superoxide dismutase (SOD) (1-50 units/mL; up to 90%) or semicarbazide (0.5-5 mM; up to 80%) and by EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which detected the formation of the DMPO-(*)OH adduct as a decomposition product from the DMPO-O(2)(*)(-) adduct. Accordingly, oxygen uptake by AA is accelerated upon addition of xanthine/xanthine oxidase, a well-known enzymatic source of O(2)(*)(-) radicals. Under Fe(II)EDTA catalysis, SOD (<50 units/mL) had little effect on the oxygen uptake curve or on the EPR spectrum of AA/DMPO, which shows intense signals of the DMPO-(*)OH adduct and of a secondary carbon-centered DMPO adduct, attributable to the AA(*) enoyl radical. In the presence of iron, simultaneous (two) electron transfer from both Fe(II) and AA to O(2), leading directly to H(2)O(2) generation followed by the Fenton reaction is thought to take place. Aminoacetone was also found to induce dose-dependent Fe(II) release from horse spleen ferritin, putatively mediated by both O(2)(*)(-) and AA(*) enoyl radicals, and the co-oxidation of added hemoglobin and myoglobin, which may be viewed as the initial step for potential further iron release. It is thus tempting to propose that AA, accumulated in the blood and other tissues of diabetics, besides being metabolized by SSAO, may release iron and undergo spontaneous and iron-catalyzed oxidation with production of reactive H(2)O(2) and O(2)(*)(-), triggering pathological responses. It is noteworthy that noninsulin-dependent diabetes has been frequently associated with iron overload and oxidative stress.

Selective inhibition of FAD and copper-dependent amine oxidases by N-acetyl pyrazole derivatives.

Selective inhibition of FAD and copper-dependent amine oxidases by N-acetyl pyrazole derivatives.

Characterization of a Model Compound for the Lysine Tyrosylquinone Cofactor of Lysyl Oxidase

We characterized a model compound for the lysine tyrosylquinone (LTQ) cofactor of lysyl oxidase which is one of the mammalian copper-dependent amine oxidases. The model compound, 4-butylamino-5-methyl-o-quinone, was prepared from n-butylamine and 4-methylcatechol by the oxidation with sodium iodate and characterized by spectroscopic analyses. The absorption maximum at 494 nm is consistent with that of lysyl oxidase. The model compound was capable of deaminating benzylamine to benzaldehyde at 37°C in buffered aqueous acetonitrile. The aldehyde production was markedly elevated in the presence of the Cu(II)-EDTA complex but inhibited by free Cu(II). The catalytic cycle was observed at pH 10 in the presence of Cu(II), and the pH activity profile showed a broad optimum at about pH 9.0. In the presence of β-aminopropionitrile and upon deoxygenation with N2 aldelyde, production was decreased. The important features of the reaction were consistent with the enzymatic reaction.

Copper(I) complexes with N,O-donor Schiff base ligands related to intermediate forms of the TPQ cofactor in amine oxidases

Neutral complexes (PPh3)2Cu(L) were obtained from (PPh3)2Cu(NO3) and the deprotonated forms of the three Schiff base ligands 2-(benzylideneimino)phenol (BimOH), 4-(benzylideneimino)resorcinol (Bim(OH)2) and N-(2,4-dihydroxy-5-isopropylphenyl)acetamide (DipaH3). The ligands were designed to model aminated intermediate forms of the topaquinone (TPQ) cofactor in the enzymatic cycle of copper-dependent amine oxidases. The ligands L are coordinated to the metal centers through imine-N and phenolate-O donor atoms to form five-membered chelate rings, in contrast to the six-membered chelate rings of salen-type Schiff base ligands. Compound (Bim(OH)O)Cu(PPh3)2·0.5CH3OH was structurally analyzed, it exhibits intramolecular aryl–aryl interactions between the ligands and intermolecular hydrogen bonds involving the free phenolic hydroxyl substituent, the coordinated phenolate oxygen atom and the methanol solvate molecule.

Lysyl oxidase: properties, regulation and multiple functions in biology.

Lysyl oxidase (LO) is a copper-dependent amine oxidase that plays a critical role in the biogenesis of connective tissue matrices by crosslinking the extracellular matrix proteins, collagen and elastin. Levels of LO increase in many fibrotic diseases, while expression of the enzyme is decreased in certain diseases involving impaired copper metabolism. While the three-dimensional structure of the enzyme is not yet available, many of its physical-chemical properties, its novel carbonyl cofactor, and its catalytic mechanism have been described. Lysyl oxidase is synthesized as a preproprotein, secreted as a 50 kDa, N-glycosylated proenzyme and then proteolytically cleaved to the 32 kDa, catalytically active, mature enzyme. Within the past decade, the gene encoding LO has been cloned, facilitating investigations of the regulation of expression of the enzyme in response to diverse stimuli and in numerous disease states. Transforming growth factor-beta, platelet-derived growth factor, angiotensin II, retinoic acid, fibroblast growth factor, altered serum conditions, and shear stress are among the effectors or conditions that regulate LO expression. New, LO-like genes have also been identified and cloned, suggesting the existence of a multigene family. It has also become increasingly evident that LO may have other important biological functions in addition to its role in the crosslinking of elastin and collagen in the extracellular matrix.

Kinetics and stereospecificity of the lysyl oxidase reaction

The structural specificity of amine oxidation by lysyl oxidase was investigated using kinetic and NMR spectroscopic analyses. Substrate efficiency increased with increasing molecular distance from the alpha-carbon of the aromatic moiety substituted on the aliphatic chains of a series of primary amines. The p-hydroxyl substituent of p-hydroxybenzylamine significantly increased kcat over that of benzylamine, whereas this was not the case when tyramine and phenethylamine were compared. Direct spectrophotometric measurement of p-hydroxybenzaldehyde formation yielded burst kinetics, the second, slower phase of which was eliminated under anaerobic conditions. Thus, enzyme reoxidation is the more rate-limiting of the two half-reactions catalyzed with this substrate by this ping-pong enzyme. 1H NMR spectroscopy of the alcohol reductively derived from the aldehyde product of the lysyl oxidase-catalyzed oxidation of deuterated tyramine indicated that the pro-S but not the pro-R alpha-deuteron was catalytically abstracted. Moreover, lysyl oxidase catalyzed solvent exchange of protons at the C-2 position. Such stereospecificity and proton exchange uniquely differentiates lysyl oxidase from all but an aortic semicarbazide-sensitive amine oxidase among the pro-S-specific copper-dependent amine oxidases analyzed thus far.

Ruthenium(II) coordination to a model for the topasemiquinone cofactor of amine oxidases. Resolution of1H and99,101Ru EPR hyperfine structure

The paramagnetic complex (L2–˙) RuII(bpy)2, where L2–˙= 5-methyl-2-oxido-1,4-benzosemiquinone and bpy = 2,2′-bipyridine, was studied because the quinonoid form LH is a model of the topa-quinone (TPQ) cofactor of certain copper-dependent amine oxidases. For the metal complex the potential ortho- and para-quinonoid character of the LHn system was probed by high resolution EPR spectroscopy of the semiquinone state which could be obtained through oxidation of the basic catecholate form.

Functional analysis of the 5' flanking domain of the LOXL4 gene in head and neck squamous cell carcinoma cells

Lysyl oxidases are a family of five copper-dependent amine oxidases including LOX, LOXL, LOXL2, LOXL3 and LOXL4. LOX and LOXL are essential for the assembly and maintenance of extracellular matrixes. LOXL2, LOXL3 and LOXL4, secreted and active enzymes, were also noted in association with diverse tumor types. We have recently reported overexpression of the LOXL4 mRNA and protein and a close relation of LOXL4 with the pathogenesis of head and neck squamous cell carcinomas (HNSCC). In this study, we analyzed the organization of the LOXL4 gene and addressed the regulatory mechanisms responsible for the overexpression. We demonstrated de novo transcription of the LOXL4 gene in HNSCC, but not in normal squamous epithelial cells. Analysis of the consecutive promoter region spanning positions -960 to -1 identified binding sites for several transcription factors. Promoter constructs containing selected specific promoter regions and consensus binding sites exhibited significantly increased reporter gene activity in HNSCC cells, but not in normal epithelial cells in transient coexpression experiments. The activity profiles of some of these constructs were similar in both cell types indicating that elements of the basic transcriptional regulatory mechanisms remained intact in HNSCC cells. DNA-binding experiments demonstrated that nuclear extracts from HNSCC cells have increased binding activity to the TATA (-25) and the SP1 (-181) sites compared to normal epithelial cells, suggesting that these transcription factors are involved in the upregulation of LOXL4 gene expression in HNSCC.

Specific detection of quinoproteins by redox-cycling staining.

Quinones and related quinonoid substances catalyze redox cycling at an alkaline pH in the presence of excess glycine as reductant. With nitroblue tetrazolium and oxygen present there is concomitant reduction of the tetrazolium to formazan. This property of quinonoid compounds is used for the specific staining of quinoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. The dopa-containing vitelline proteins and the 6-hydroxydopa-containing bovine serum amine oxidase are stained with the nitroblue tetrazolium/glycinate reagent. Also, the mammalian quinoproteins, diamine oxidase and lysyl oxidase, purported to contain pyrroloquinoline quinone, tested positive in this procedure. No quinonoid components were detected in three putative pyrroloquinoline quinone-containing quinoproteins, dopamine beta-hydroxylase, lipoxygenase, and peptidylglycine-amidating monoxygenase. Redox-cycling staining therefore confirms the presence of covalently bound quinones in the copper-dependent amine oxidases, but not in two putative quinoprotein oxygenases. Clarification of the biological significance of quinolation should be facilitated by identification of quinoproteins using this approach.

Gas chromatographic determination of N-acetylisoputreanine-γ-lactam, a unique catabolite of N 1-acetylspermidine

A capillary gas chromatographic method with nitrogen—phosphorus detection for the determination of N-acetylisoputreanine-γ-lactam (acisoga) in urine is described. The method was validated by comparing the results with those given by an isotope dilution mass fragmentographic method. Making use of specific inhibitors for copper-dependent amine oxidase and polyamine oxidase in rats, it was demonstrated that acisoga is formed by oxidative deamination of N 1-acetylspermidine by the former enzyme. Moreover, acisoga is not a substrate for pig liver polyamine oxidase. Increased concentrations of acisoga, relative to N 1-acetylspermidine, in urines of patients with non-Hodgkin's lymphoma indicated that this conversion diminishes the sensitivity of N 1-acetylspermidine as a marker for (tumour) cell death.

Heat shock stimulates polyamine oxidation by two distinct mechanisms in mammalian cell cultures.

Heat shock stimulates both exogenous and endogenous polyamine oxidation in mammalian cells, but by distinct biochemical mechanisms. Exogenously added polyamines are oxidized in serum via the temperature-dependent activation of a single class of enzymes, the copper-dependent amine oxidases. Endogenous polyamines undergo a two-step reaction sequence involving acetylation by a heat-inducible acetyltransferase and subsequent oxidation by a constitutively expressed, flavin-dependent polyamine oxidase. In both instances, polyamine oxidation generates hydrogen peroxide and reactive aldehydes which influence cell viability as demonstrated by inhibitor studies. Aminoguanidine, an inhibitor of the copper-dependent amine oxidases, confers protection to cells during either a severe 43 degrees C heat shock or a relatively nontoxic heat stress followed by incubation at 37 degrees C, all in the presence of exogenous spermidine. Specific inhibition of the endogenous polyamine oxidase will also confer partial survival protection after heat shock, but only in cultures that have been previously depleted of cellular glutathione. These data confirm that hyperthermic stress can generate an oxidative stress in mammalian cells via induction of polyamine oxidation. Further, through distinct extracellular and intracellular mechanisms, these temperature-dependent polyamine oxidation reactions can modulate cell viability.

Inhibition of copper-dependent amine oxidases by some hydrazides of pyrrol-1-ylbenzoic and pyrrol-1-ylphenylacetic acids.

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.

Copper-dependent amine oxidases in angiogenesis induced by prostaglandin E 1 (PGE 1)

Copper-dependent amine oxidases in angiogenesis induced by prostaglandin E 1 (PGE 1)

Reaction pathway of bovine aortic lysyl oxidase.

The catalysis of amine oxidation by lysyl oxidase has been probed to assess for the likely order of substrate binding and product release and to discriminate between mechanistic alternatives previously proposed for other copper-dependent amine oxidases using molecular oxygen as a substrate. Lineweaver-Burk plots revealed a pattern of parallel lines when the oxidation of n-butylamine was followed at different fixed concentrations of oxygen consistent with a "ping-pong" kinetic mechanism in which the aldehyde is produced and released before the binding of oxygen, the second substrate. Initial burst experiments revealed the ability of lysyl oxidase to form and release n-butyraldehyde in amounts stoichiometric with functional active site content in the absence of oxygen, consistent with the ping-pong kinetics obtained. Reciprocal plots of n-butylamine oxidation in the presence of fixed concentrations of the reaction products were consistent with a Uni Uni Uni Bi ping-pong kinetic mechanism with the aldehyde being the first, H2O2 the second, and ammonia the last departing product. Moreover, spectral studies of the oxidation of p-hydroxybenzylamine by lysyl oxidase indicated that the enzyme does not process the amine substrate to a noncovalently bound p-hydroxybenzaldimine intermediate subsequently to be hydrolyzed to p-hydroxybenzaldehyde. The kinetic mechanism of lysyl oxidase thus appears to be similar to those described for diamine oxidase and pig plasma monoamine oxidase.

Monoamine oxidase activities in brown adipose tissue of the rat: Some properties and subcellular distribution

Amine oxidase activity towards 5-hydroxytryptamine (5HT), tyramine (TYR), 2-phenylethylamine (PEA) and benzylamine (BZ) was studied in homogenates of interscapular brown adipose tissue of the rat. By the use of clorgyline, an irreversible inhibitor of MAO, it was established that 5HT was deaminated solely by MAO-A, and TYR and PEA mainly by MAO-A and clorgyline-resistant semicarbazide-sensitive amine oxidase (CRAO). BZ appeared to be oxidized almost entirely by CRAO. A very small amount of MAO-B activity was detectable with PEA and BZ as substrates. A variety of amines, amino acids and known amine oxidase inhibitors were tested for their ability to inhibit the deamination of BZ by CRAO. BZ metabolism by the enzyme was not affected by any secondary amines, unlike enzymes of the flavin type, but it was inhibited by carbonyl reagents, like the pyridoxal phosphate and copper-dependent amine oxidases described in plasma and connective tissue. Unlike these enzymes, however, CRAO in brown adipose tissue was resistant to KCN and unaffected by the amines, histamine, mescaline and some polyamines but it was inhibited by cuprizone. It was found to have a low K m (<5 μM) for BZ and showed the greatest similarity to a clorgyline-resistant enzyme described in rat blood vessels. Cell fractionation studies revealed that CRAO, being associated with the particulate fractions, was mainly membrane-bound. The distribution of CRAO activity between various cell fractions was different from that of the mitochondrial enzymes assayed and was more like that of either the plasma membrane or microsomal enzymes. When microsomal and plasma membrane vesicles were separated CRAO activity appeared distributed equally between the two fractions, suggesting that the enzyme may have a dual location within the cell. The specific activity of CRAO was higher in brown adipose tissue from obese animals than in tissue from lean animals. The significance of these findings is discussed in relation to the possible physiological function of this enzyme.

Diamine oxidase from pig kidney: new purification method and amino acid composition.

Several methods for the isolation of apparently homogeneous pig kidney diamine oxidase have been reported in recent years (1-7), but these procedures allow to obtain only little amounts of material making very difficult the study of the molecular properties of the enzyme. Drawing useful indication from the purification procedures previously reported, we were able to set up a new method which allows to obtain homogeneous enzyme samples in high yield and with good reproducibility. This procedure allowed to determine with greater accuracy the molecular weight of the enzyme that resulted to be 170,000 daltons by gel chromatography and 145,000 by ultracentrifuge. The enzyme is composed of two apparently identical subunits and contains two copper atoms per dimer. The amino acid composition of the protein has been also worked out and found similar to those already reported for other copper dependent amine oxidases. Pig kidney diamine oxidase is a glycoprotein containing about 20% sugars by weight.

Copper and amine oxidases in connective tissue metabolism.

That copper is an essential biocatalyst has been recognized for nearly a half century but its critical role in connective tissue metabolism has come into focus only in recent years. It is now clear that copper plays a role in the crosslinking of the two important connective tissue proteins, elastin and collagen. The gross pathology that results from this copper deficiency is most dramatic when it occurs in large arteries and results in spontaneous and massive hemorrhage. Because of this unique pathology and the importance of a sound vasculature to human health, our research has been focused on the aorta. The effect of copper deficiency on the integrity of collagen and elastin and the activity of the amine oxidases in this organ has been investigated. This paper summarizes earlier observations and presents recent results relating to a copper-dependent amine oxidase which plays a key role in the crosslinking process.