Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases.

Four substrate analogs, 4-(2-naphthyloxy)-2-butyn-1-amine (1), 1,4-diamino-2-chloro-2-butene (2), 1,6-diamino-2,4-hexadiyne (3), and 2-chloro-5-phthalimidopentylamine (4) have been tested as inhibitors against mammalian, plant, bacterial, and fungal copper-containing amine oxidases: bovine plasma amine oxidase (BPAO), equine plasma amine oxidase (EPAO), pea seedling amine oxidase (PSAO), Arthrobacter globiformis amine oxidase (AGAO), Escherichia coli amine oxidase (ECAO), and Pichia pastoris lysyl oxidase (PPLO). Reactions of 1,4-diamino-2-butyne with selected amine oxidases were also examined. Each substrate analog contains a functional group that chemical precedent suggests could produce mechanism-based inactivation. Striking differences in selectivity and rates of inactivation were observed. For example, between two closely related plasma enzymes, BPAO is more sensitive than EPAO to 1 and 3, while the reverse is true for 2 and 4. In general, inactivation appears to arise in some cases from TPQ cofactor modification and in other cases from alkylation of protein residues in a manner that blocks access of substrate to the active site. Notably, 1 completely inhibits AGAO at stoichiometric concentrations and is not a substrate, but is an excellent substrate of PSAO and inhibition is observed only at very high concentrations. Structural models of 1 in Schiff base linkage to the TPQ cofactor in AGAO and PSAO (for which crystal structures are available) reveal substantial differences in the degree of interaction of bound 1 with side-chain residues, consistent with the widely divergent activities. Collectively, these results suggest that the development of highly selective amine oxidase inhibitors is feasible.     

Inhibition of bovine plasma amine oxidase by 1,4-diamino-2-butenes and -2-butynes

Bovine plasma amine oxidase (BPAO) was previously shown to be irreversibly inhibited by propargylamine and 2-chloroallylamine. 1,4-Diamine versions of these two compounds are here shown to be highly potent inactivators, with IC50 values near 20 microM. Mono-N-alkylation or N,N-dialkylation greatly lowered the inactivation potency in every case, whereas the mono-N-acyl derivatives were also weaker inhibitors and enzyme activity was recoverable. The finding that the bis-primary amines 1,4-diamino-2-butyne (a known potent inhibitor of diamine oxidases) and Z-2-chloro-1,4-diamino-2-butene are potent inactivators of BPAO is suggestive of unexpected similarities between plasma amine oxidase and the diamine oxidases and implies that it may be unwise to attempt to develop selective inhibitors of diamine oxidase using a diamine construct.     

Reversible inactivation of bovine plasma amine oxidase by cysteamine and related analogs

Cysteamine (1) was reported many years ago to reversibly inhibit lentil seedling amine oxidase, through the formation of a complex with thioacetaldehyde, the turnover product of 1. Herein, cysteamine (1) and its analogs 2-(methylamino)ethanethiol (3) and 3-aminopropanethiol (6) were found to be reversible inhibitors of bovine plasma amine oxidase (BPAO), but 2-(methylthio)ethylamine (7) was determined to be a weak irreversible inhibitor of BPAO. Based on our results, indicating the necessity of a sulfhydryl-amine for reversible inactivation of BPAO, the failure of inhibited BPAO to recover activity after gel filtration, the first-order kinetics of activity recovery upon dialysis, and 2,4,6-trihydroxyphenylalanine quinine (TPQ) cofactor transformation which indicated from the results of phenylhydrazine titration and substrate protection, we propose a mechanism for the reversible inactivation of BPAO by 1 involving the formation of a cofactor adduct, thiazolidine, between BPAO and 1.     

Inactivation of copper-containing amine oxidases by turnover products.

For bovine serum amine oxidase, two different mechanisms of substrate-induced inactivation have been proposed. One consists of a slow oxidation by H2O2 of a conserved residue in the reduced enzyme after the fast turnover phase [Pietrangeli, P., Nocera, S., Fattibene, P., Wang, X.T., Mondovì, B. & Morpurgo, L. (2000) Biochem. Biophys. Res. Commun.267, 174-178] and the other of the oxidation by H2O2 of the dihydrobenzoxazole in equilibrium with the product Schiff base, during the catalytic cycle [Lee, Y., Shepard, E., Smith, J., Dooley, D.M. & Sayre, L.M. (2001) Biochemistry40, 822-829]. To discriminate between the two mechanisms, the inactivation was studied using Lathyrus cicera (red vetchling) amine oxidase. This, in contrast to bovine serum amine oxidase, formed the Cu+-semiquinolamine radical with a characteristic UV-vis spectrum when oxygen was exhausted by an excess of any tested amine in a closed cuvette. The inactivation, lasting about 90 min, was simultaneous with the radical decay and with the formation of a broad band (shoulder) at 350 nm. No inactivation occurred when a thousand-fold excess of amine was rapidly oxidized in an L. cicera amine oxidase solution stirred in open air. Thus, the inactivation is a slow reaction of the reduced enzyme with H2O2, following the turnover phase. Catalase protected L. cicera amine oxidase from inactivation. This effect was substrate-dependent, varying from full protection (benzylamine) to no protection (putrescine). In the absence of H2O2, a specific inactivating reaction, without formation of the 350 nm band, was induced by some aldehydes, notably putrescine. Some mechanisms of inactivation are proposed.     

Spectroscopic observation of intermediates formed during the oxidative half-reaction of copper/topa quinone-containing phenylethylamine oxidase.

The catalytic reaction of copper/topa quinone (TPQ) containing amine oxidase consists of the initial, well-characterized, reductive half-reaction and the following, less studied, oxidative half-reaction. We have analyzed the oxidative half-reaction catalyzed by phenylethylamine oxidase from Arthrobacter globiformis (AGAO) by rapid-scan stopped-flow measurements. Upon addition of dioxygen to the substrate-reduced AGAO at pH 8.2, the absorption bands derived from the semiquinone (TPQ(sq)) and aminoresorcinol forms of the TPQ cofactor disappeared within the dead time (<1 ms) of the measurements, indicating that the reaction of the substrate-reduced enzyme with dioxygen is very rapid. Concomitantly, an early intermediate exhibiting an absorption band at about 410 nm was formed, which then decayed with a rate constant of 390 +/- 50 s(-1). This intermediate was detected more prominently in the reaction in D2O buffer (pD 8.1) and was assigned to a Cu(II)-peroxy species. The assignment was based on the observation that addition of H2O2 to the substrate-reduced AGAO under anaerobic conditions led to the formation of a new band at about 415 nm, accompanied by partial quenching of absorption bands derived from TPQ(sq). Other intermediates exhibiting absorption bands at about 310 and 340 nm were also observed in the oxidative half-reaction. Kinetics of the disappearance of these latter bands did not correspond with that of the Cu(II)-peroxy band at 410 nm but did well with that of the increase of the 480 nm absorption band due to the reoxidized TPQ. Rapid increase of the absorption in the 320-370 nm region was also observed for the reaction of the substrate-reduced, Ni-substituted enzyme with dioxygen. On the basis of these results, a possible mechanism is proposed for the oxidative half-reaction of the bacterial copper amine oxidase.     

Cyanide as a copper-directed inhibitor of amine oxidases: implications for the mechanism of amine oxidation

 The interactions of five copper-containing amine oxidases with substrates and substrate analogues in the presence of the copper ligands cyanide, azide, chloride, and 1,10-phenanthroline have been investigated. While cyanide inhibits, to varying degrees, the reaction of phenylhydrazine with porcine kidney amine oxidase (PKAO), porcine plasma amine oxidase (PPAO), bovine plasma amine oxidase (BPAO), and pea seedling amine oxidase (PSAO), it enhances the reaction of Arthrobacter P1 amine oxidase (APAO) with this substrate analogue. This indicates that cyanide exerts an indirect effect on topa quinone (TPQ) reactivity via coordination to Cu(II) rather than through cyanohydrin formation at the TPQ organic cofactor. Moreover, cyanide binding to the mechanistically relevant TPQ^• semiquinone form of substrate-reduced APAO and PSAO was not observable by EPR or resonance Raman spectroscopy. Hence, cyanide most likely inhibits enzyme reoxidation by binding to Cu(I) and trapping the Cu(I)-TPQ^• form of amine oxidases, and thus preventing the reaction of O₂ with Cu(I). In contrast, ligands such as azide, chloride, and 1,10-phenanthroline, which preferentially bind to Cu(II), inhibit by stabilizing the aminoquinol Cu(II)-TPQ_red redox state, which is in equilibrium with Cu(I)-TPQ^•. Received: 12 December 1996 / Accepted: 20 March 1997     

An important lysine residue in copper/quinone-containing amine oxidases

The interaction of xenon with copper/6-hydroxydopa (2,4,5-trihydroxyphenethylamine) quinone (TPQ) amine oxidases from the plant pulses lentil (Lens esculenta) and pea (Pisum sativum) (seedlings), the perennial Mediterranean shrub Euphorbia characias (latex), and the mammals cattle (serum) and pigs (kidney), were investigated by NMR and optical spectroscopy of the aqueous solutions of the enzymes. (129)Xe chemical shift provided evidence of xenon binding to one or more cavities of all these enzymes, and optical spectroscopy showed that under 10 atm of xenon gas, and in the absence of a substrate, the plant enzyme cofactor (TPQ), is converted into its reduced semiquinolamine radical. The kinetic parameters of the analyzed plant amine oxidases showed that the k(c) value of the xenon-treated enzymes was reduced by 40%. Moreover, whereas the measured K(m) value for oxygen and for the aromatic monoamine benzylamine was shown to be unchanged, the K(m) value for the diamine putrescine increased remarkably after the addition of xenon. Under the same experimental conditions, the TPQ of bovine serum amine oxidase maintained its oxidized form, whereas in pig kidney, the reduced aminoquinol species was formed without the radical species. Moreover the k(c) value of the xenon-treated pig enzyme in the presence of both benzylamine and cadaverine was shown to be dramatically reduced. It is proposed that the lysine residue at the active site of amine oxidase could be involved both in the formation of the reduced TPQ and in controlling catalytic activity.     

Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2-butynamines: evidence for a new mode of inactivation

A series of compounds derived from a previously identified substrate analogue of copper amine oxidases (CuAOs) (Shepard et al. (2002) Eur. J. Biochem. 269, 3645-3658) has been screened against six different CuAOs with a view to designing potent and selective inhibitors. The substrate analogues investigated were 4-(1-naphthyloxy)-2-butyn-1-amine, 4-(2-methylphenoxy)-2-butyn-1-amine, 4-(3-methylphenoxy)-2-butyn-1-amine, 4-(4-methylphenoxy)-2-butyn-1-amine, and 4-phenoxy-2-butyn-1-amine. These compounds were screened against equine plasma amine oxidase (EPAO), Pisum sativum amine oxidase (PSAO), Pichia pastoris lysyl oxidase (PPLO), bovine plasma amine oxidase (BPAO), human kidney diamine oxidase (KDAO), and Arthrobacter globiformis amine oxidase (AGAO) to examine the effect of different substituent groups on potency. Despite the similar structures of the 4-aryloxy analogues evaluated, striking differences in potency were observed. In addition, crystal structures of AGAO derivitized with 4-(2-naphthyloxy)-2-butyn-1-amine and 4-(4-methylphenoxy)-2-butyn-1-amine were obtained at a resolution of 1.7 A. The structures reveal a novel and unprecedented reaction mechanism involving covalent attachment of the alpha,beta-unsaturated aldehyde turnover product to the amino group of the reduced 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. Collectively, the structural and inhibition results support the feasibility of designing selective mechanism-based inhibitors of copper amine oxidases.     

Rates of oxygen and hydrogen exchange as indicators of TPQ cofactor orientation in amine oxidases.

This study presents the first detailed examination by resonance Raman (RR) spectroscopy of the rates of solvent exchange for the C5 and C3 positions of the TPQ cofactor in several wild-type copper-containing amine oxidases and mutants of the amine oxidase from Hansenula polymorpha (HPAO). On the basis of crystal structure analysis and differing rates of C5 [double bond] O and C3 [bond] H exchange within the enzyme systems, but equally rapid rates of C5 [double bond] O and C3 [bond] H exchange in a TPQ model compound, it is proposed that these data can be used to determine the TPQ cofactor orientation within the active site of the resting enzyme. A rapid rate of C5 [double bond] O exchange (t(1/2) < 30 min) and a slow (t(1/2) = 6 h) to nonexistent rate of C3 [bond] H exchange was observed for wild-type HPAO, the amine oxidase from Arthrobacter globiformis, pea seedling amine oxidase at pH 7.1, and the E406Q mutant of HPAO. This pattern is ascribed to a productive TPQ orientation, with the C5 [double bond] O near the substrate-binding site and the C3 [bond] H near the Cu. In contrast, a slow rate of C5 [double bond] O exchange (t(1/2) = 1.6-3.3 h) coupled with a fast rate of C3 [bond] H exchange (t(1/2) < 30 min) was observed for the D319E and D319N catalytic base mutants of HPAO and for PSAO at pH 4.6 (t(1/2) = 4.5 h for C5 [double bond] O exchange). This pattern identifies a flipped orientation, involving 180 degrees rotation about the C alpha-C beta bond, which locates the C3 [bond] H near the substrate-binding site and the C5 double bond] O near the Cu. Finally, fast rates of both C5 [double bond] O and C3 [bond] H exchange (t(1/2) < 30 min) were observed for the amine oxidase from Escherichia coli and the N404A mutant of HPAO, suggesting a mobile cofactor, with multiple TPQ orientations between productive and flipped. These results demonstrate that opposing sides of the TPQ ring possess different degrees of solvent accessibility and that the rates of C5 [double bond] O and C3 [bond] H exchange can be used to predict the TPQ cofactor orientation in the resting forms of these enzymes.     

Reductive trapping of substrate to bovine plasma amine oxidase

Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O2 to H2O2. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation between a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of [14C]benzylamine and [3H]NaCNBH3. The use of the relatively weak reductant, NaCNBH3, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH3 in the presence of substrate, leading to the incorporation of 1 mol of [14C]benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with [3H]NaCNBH3, analogous to an earlier study with [3H]NaCNBH4 (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.     

Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302

Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.     

Amine Oxidase from Lentil Seedlings: Energetic Domains and Effect of Temperature on Activity

Copper/TPQ amine oxidases from mammalian and plant sources have shown many differences in substrate specificity and molecular properties. In this work the activity of lentil seedling amine oxidase was followed at various temperatures in 100 mM potassium phosphate buffer, pH 7, using benzylamine as substrate. The discontinuous Arrhenius plot of lentil amine oxidase showed two distinct phases with a jump between them. Thermal denaturation of the enzyme, using differential scanning calorimetry under the same experimental conditions, showed a transition at the same temperature ranges in the absence of substrate, indicating the occurrence of conformational changes, with an enthalpy change of about 175.9 kJ/mole. The temperature-induced changes of the activity of lentil amine oxidase are compared with those of bovine serum amine oxidase (taken from the literature).     

Stopped-flow spectrophotometric characterization of enzymic reaction intermediates in the anaerobic reduction of pig-plasma benzylamine oxidase by amine substrates.

Reduction of benzylamine oxidase by p-methoxybenzylamine under anaerobic conditions leads to biphasic absorbance changes at 470 nm. These reflect the intermediate formation of an enzyme substrate complex with spectral properties different from those of native enzyme and fully reduced enzyme. The spectrally modified enzyme-substrate complex exhibits a broad difference absorption band centered around 360 nm. The transient accumulation of this intermediate during reaction can be conveniently followed by stopped-flow techniques at wavelengths between 320 and 360 nm, where contributions from the subsequent reduction of the enzymic 470-nm chromophore are of minor significance. 2. Analogous intermediates exhibiting similar absorption spectra seem to be formed on reduction of the enzyme by benzylamine and other amine substrates which were tested. Substitution of benzylamine as the reducing substrate by [alpha, alpha-2H]benzylamine results in a decreased accumulation of the spectrally modified intermediate. This indicates that its formation is preceded by deprotonation of the alpha-carbon of the amine substrate. 3. Circular dichroism spectra of benzylamine oxidase exhibit a positive band at 360 nm, lending support to the previous conclusion that benzylamine oxidase is a pyridoxal enzyme. Formation of the spectrally modified enzyme-substrate complex then most likely reflects the prototropic shift converting an amine-pyridoxal Schiff-base obtained by rapid pre-equilibration between enzyme and substrate into an aldehyde-pyridoxamine Schiff-base.     

Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[alpha-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the alpha-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the alpha-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[alpha-2H1]dopamine, preferably breaking the C-H bond rather than the C-2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.     

Molecular mode of interaction of plant amine oxidase with the mechanism-based inhibitor 2-butyne-1,4-diamine.

2-Butyne-1,4-diamine (DABI) is a mechanism-based inhibitor of copper-containing plant amine oxidases; the number of turnovers that leads to enzyme inactivation is approximately 20. The product of DABI oxidation is a very reactive aminoallene that reacts with an essential nucleophilic group at the enzyme active site, forming a covalently bound pyrrole and producing an inactive enzyme. The inactivated enzyme shows a new absorption maximum at 295 nm and gives coloured derivatives with p-dimethylaminobenzaldehyde and p-dimethylaminocinnamaldehyde that are spectrally similar to the products of pyrrole treated with the above reagents. Resonance Raman spectra of the p-dimethylaminobenzaldehyde adduct of pyrrole and the inactivated enzyme show very high degree of similarity, supporting the idea that the product of inactivation is indeed a bound pyrrole. The bound pyrrole is formed already in the anaerobic step of the reaction, while the topa semiquinone radical is not affected, as shown by the EPR and stopped-flow absorption measurements. Peptides containing the DABI binding site were obtained by proteolysis of inactivated enzyme, isolated by HPLC and analysed by amino acid sequencing and MS. The crystal structure of the amine oxidase from pea has been determined; inhibition is caused mainly by the highly reactive DABI product, 4-amino-2-butynal, binding to a nucleophilic residue at the entrance to the substrate channel. As other DABI labelled peptides were also found and no free DABI product was detected by MS after complete inhibition of the enzyme, it is likely that the DABI product binds also to other solvent exposed nucleophilic residues on the enzyme surface.     

An unexpected formation of the spectroscopic Cu(I)-semiquinone radical by xenon-induced self-catalysis of a copper quinoprotein

Plant copper/quinone amine oxidases are homodimeric enzymes containing Cu(II) and a quinone derivative of a tyrosyl residue (2,4,5-trihydroxyphenylalanine, TPQ) as cofactors. These enzymes catalyze the oxidative deamination of primary amines by a classical ping-pong mechanism, i.e. two distinct half-reactions, enzyme reduction by substrate followed by its re-oxidation by molecular oxygen. In the first half-reaction two forms of the reduced TPQ have been observed, the colorless Cu(II)-aminoquinol and the yellow Cu(I)-semiquinolamine radical so that this enzyme may be referred to as a "protein-radical enzyme". The interaction of xenon, in aqueous solutions, with the copper/TPQ amine oxidase from lentil (Lens esculenta) seedlings has been investigated by NMR and optical spectroscopy. NMR data indicate that xenon binds to the protein. Under 10 atm gaseous xenon and in the absence of substrates more than 60% native enzyme is converted into Cu(I)-semiquinolamine radical species, showing for the first time that both monomers in the dimer can generate the radical. Under the same experimental conditions the copper-free lentil enzyme is able to generate an intermediate absorbing at about 360 nm, which is assigned to the product Schiff base quinolaldimine which, to the best of our knowledge, has never been observed during the catalytic mechanism of plant amine oxidases. A possible role of the lysine residue responsible for the formation of Cu(I)-semiquinolamine and quinolaldimine, is proposed.     

Mechanism-based inactivators of plant copper/quinone containing amine oxidases

Copper/quinone amine oxidases contain Cu(II) and the quinone of 2,4,5-trihydroxyphenylalanine (topaquinone; TPQ) as cofactors. TPQ is derived by post-translational modification of a conserved tyrosine residue in the protein chain. Major advances have been made during the last decade toward understanding the structure/function relationships of the active site in Cu/TPQ amine oxidases using specific inhibitors. Mechanism-based inactivators are substrate analogues that bind to the active site of an enzyme being accepted and processed by the normal catalytic mechanism of the enzyme. During the reaction a covalent modification of the enzyme occurs leading to irreversible inactivation. In this review mechanism-based inactivators of plant Cu/TPQ amine oxidases from the pulses lentil (Lens esculenta), pea (Pisum sativum), grass pea (Lathyrus sativus) and sainfoin (Onobrychis viciifolia,) are described. Substrates forming, in aerobiotic and in anaerobiotic conditions, killer products that covalently bound to the quinone cofactor or to a specific amino acid residue of the target enzyme are all reviewed.     

Cyanide as a copper and quinone-directed inhibitor of amine oxidases from pea seedlings (<Emphasis Type="Italic">Pisum sativum</Emphasis>) and<Emphasis Type="Italic"> Arthrobacter globiformis</Emphasis>: evidence for both copper coordination and cyanohydrin derivatization of the quinone cofactor

The interactions of cyanide with two copper-containing amine oxidases (CuAOs) from pea seedlings (PSAO) and the soil bacterium Arthrobacter globiformis (AGAO) have been investigated by spectroscopic and kinetic techniques. Previously, we rationalized the effects of azide and cyanide for several CuAOs in terms of copper coordination by these exogenous ligands and their effects on the internal redox equilibrium TPQ_amr-Cu(II)TPQ_sq-Cu(I). The mechanism of cyanide inhibition was proposed to occur through complexation to Cu(I), thereby directly competing with O₂ for reoxidation of TPQ. Although cyanide readily and reversibly reacts with quinones, no direct spectroscopic evidence for cyanohydrin derivatization of TPQ has been previously documented for CuAOs. This work describes the first direct spectroscopic evidence, using both model and enzyme systems, for cyanohydrin derivatization of TPQ. K_d values for Cu(II)-CN^– and Cu(I)-CN^–, as well as the K_i for cyanide inhibition versus substrate amine, are reported for PSAO and AGAO. In spite of cyanohydrin derivatization of the TPQ cofactor in these enzymes, the uncompetitive inhibition of amine oxidation is determined to arise almost exclusively through CN^– complexation of Cu(I).Abbreviations AGAO Arthrobacter globiformis amine oxidase - APAO Arthrobacter P1 amine oxidase - APT attached proton test - BPAO bovine plasma amine oxidase - CuAO quinone-copper containing amine oxidase - LTQ lysyl tyrosylquinone - MAO monoamine oxidase - PKAO porcine kidney amine oxidase - PPAO porcine plasma amine oxidase - PSAO pea seedling amine oxidase - TPQ 2,4,5-trihydroxyphenylalaninequinone - TPQ_amr TPQ aminoresorcinol - TPQ_imq TPQ iminoquinone - TPQ_ox TPQ oxidized - TPQ_sq TPQ semiquinone - WT wild-typeE.M. Shepard and G.A. Juda contributed equally to this workThis revised version was published online in February 2004: Hansenula polymorpha was not italicised at the end of the Introduction, Equation 3 appeared twice, and the resolution of Scheme 3 was insufficient.An erratum to this article can be found at     

Structural and enzyme activity studies demonstrate that aryl substituted 2,3-butadienamine analogs inactivate Arthrobacter globiformis amine oxidase (AGAO) by chemical derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor

Copper amine oxidases (CAOs) are a family of redox active enzymes containing a 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor generated from post translational modification of an active site tyrosine residue. The Arthrobacter globiformis amine oxidase (AGAO) has been widely used as a model to guide the design and development of selective inhibitors of CAOs. In this study, two aryl 2,3-butadienamine analogs, racemic 5-phenoxy-2,3-pentadienylamine (POPDA) and racemic 6-phenyl-2,3-hexadienylamine (PHDA), were synthesized and evaluated as mechanism-based inactivators of AGAO. Crystal structures show that both compounds form a covalent adduct with the amino group of the substrate-reduced TPQ, and that the chemical structures of the rac-PHDA and rac-POPDA modified TPQ differ by the allenic carbon that is attached to the cofactor. A chemical mechanism accounting for the formation of the respective TPQ derivative is proposed. Under steady-state conditions, no recovery of enzyme activity is detected when AGAO pre-treated with rac-PHDA or rac-POPDA is diluted with excess amount of the benzylamine substrate (100-fold K(m)). Comparing the IC(50) values further reveals that the phenoxy substituent in POPDA offers an approximately 4-fold increase in inhibition potency, which can be attributed to a favourable binding interaction between the oxygen atom in the phenoxy group and the active site of AGAO as revealed by crystallographic studies. This hypothesis is corroborated by the observed >3-fold higher partition ratio of PHDA compared to POPDA. Taken together, the results presented in this study reveal the mechanism by which aryl 2,3-butadienamines act as mechanism-based inhibitors of AGAO, and the potency of enzyme inactivation could be fine-tuned by optimizing binding interaction between the aryl substituent and the enzyme active site.     

Mutation of a strictly conserved, active-site residue alters substrate specificity and cofactor biogenesis in a copper amine oxidase

The copper amine oxidases (CAOs) catalyze both the single-turnover modification of a peptidyl tyrosine to form the active-site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) and the oxidative deamination of primary amines using TPQ. The function of a strictly conserved tyrosine located within hydrogen-bonding distance to TPQ has been explored by employing site-directed mutagenesis on the enzyme from H. polymorpha to form the mutants Y305A, Y305C, and Y305F. Both Y305A and Y305C behave similarly with regard to aliphatic amine oxidase activity, showing 3-7-fold decreases in kinetic parameters relative to WT, while the more conservative substitution of Y305F results in a >100-fold decrease in kcat and >500-fold decrease in kcat/Km relative to WT for the reductive half-reaction. The oxidation of benzylamine by all three mutants is severely impaired, with very significant effects seen in the oxidative half-reaction. CAO activity was studied as a function of pH for WT and Y305A proteins. Profiles for WT-catalyzed methylamine oxidation and Y305A-catalyzed ethylamine oxidation are comparable, while profiles of Y305A-catalyzed methylamine oxidation suggest the pH-dependent build-up of an inhibitory intermediate, which was subsequently observed spectrophotometrically and is attributed to the product Schiff base. The relative effects of mutations at Y305 on catalytic turnover are, thus, concluded to be dependent on the nature of the amino acid which substitutes for tyrosine and the substrate used in amine oxidase assays. TPQ biogenesis experiments demonstrate a approximately 800-fold decrease in kobs for apo-Y305A compared to WT. Despite the strict conservation of Tyr305 in all CAOs, neither biogenesis nor catalytic turnover is abolished upon mutation of this residue. We propose an important, but nonessential, role for Tyr305 in the positioning of the TPQ precursor for biogenesis, and in the maintenance of the correct conformation for TPQ-derived intermediates during catalytic turnover.