Mechanistic studies of the yeast polyamine oxidase Fms1: kinetic mechanism, substrate specificity, and pH dependence

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N(1)-acetylspermine to yield spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. The kinetic mechanism of the enzyme has been determined with both substrates. The initial velocity patterns are ping-pong, consistent with reduction being kinetically irreversible. Reduction of Fms1 by either substrate is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with limiting rates for reduction of the enzyme of 126 and 1410 s(-1) and apparent K(d) values of 24.3 and 484 μM for spermine and N(1)-acetylspermine, respectively. The rapid phase is followed by a concentration-independent phase that is slower than turnover. The reaction of the reduced enzyme with oxygen is monophasic, with a rate constant of 402 mM(-1) s(-1) with spermine at 25 °C and 204 mM(-1) s(-1) with N(1)-acetylspermine at 4 °C and pH 9.0. This step is followed by rate-limiting product dissociation. The k(cat)/K(amine)-pH profiles are bell-shaped, with an average pK(a) value of 9.3 with spermine and pK(a) values of 8.3 and 9.6 with N(1)-acetylspermine. Both profiles are consistent with the active forms of substrates having two charged nitrogens. The pH profiles for the rate constant for flavin reduction show pK(a) values of 8.3 and 7.2 for spermine and N(1)-acetylspermine, respectively, for groups that must be unprotonated; these pK(a) values are assigned to the substrate N4. The k(cat)/K(O(2))-pH profiles show pK(a) values of 7.5 for spermine and 6.8 for N(1)-acetylspermine. With both substrates, the k(cat) value decreases when a single residue is protonated.     

Yeast Fms1 is a FAD-utilizing polyamine oxidase

In this report we show that recombinant Saccharomyces cerevisiae Fms1 protein is a polyamine oxidase that binds FAD with an FAD:Fms1 stoichiometry of 1:1. Biochemical characterization of Fms1 shows that it can oxidize spermine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine, but not spermidine. The products of spermine oxidation are spermidine and 3-aminopropanal. A kinetic analysis revealed that spermine, N(1)-acetylspermine, and N(1)-acetylspermidine are oxidized with similar efficiencies, while N(8)-acetylspermidine is a poor substrate. The data support a previous report, suggesting that Fms1 is responsible for the production of beta-alanine from spermine for the synthesis of pantothenic acid.     

Mechanistic and structural analyses of the roles of Arg409 and Asp402 in the reaction of the flavoprotein nitroalkane oxidase

The flavoprotein nitroalkane oxidase (NAO) catalyzes the oxidation of primary and secondary nitroalkanes to the corresponding aldehydes and ketones. The enzyme is a homologue of acyl-CoA dehydrogenase. Asp402 in NAO has been proposed to be the active site base responsible for removing the substrate proton in the first catalytic step; structurally it corresponds to the glutamate which acts as the base in medium chain acyl-CoA dehydrogenase. In the active site of NAO, the carboxylate of Asp402 forms an ionic interaction with the side chain of Arg409. The R409K enzyme has now been characterized kinetically and structurally. The mutation results in a decrease in the rate constant for proton abstraction of 100-fold. Analysis of the three-dimensional structure of the R409K enzyme, determined by X-ray crystallography to a resolution of 2.65 A, shows that the critical structural change is an increase in the distance between the carboxylate of Asp402 and the positively charged nitrogen in the side chain of the residue at position 409. The D402E mutation results in a smaller decrease in the rate constant for proton abstraction of 18-fold. The structure of the D402E enzyme, determined at 2.4 A resolution, shows that there is a smaller increase in the distance between Arg409 and the carboxylate at position 402, and the interaction of this residue with Ser276 is perturbed. These results establish the critical importance of the interaction between Asp402 and Arg409 for proton abstraction by nitroalkane oxidase.     

Mechanistic studies of mouse polyamine oxidase with N1,N12-bisethylspermine as a substrate

In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k(cat) value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k(cat) value decreases below a pK(a) of 9.0. The k(cat)/K(m) value for BESPM exhibits a bell-shaped pH profile, with pK(a) values of 9.8 and 10.8. These pK(a) values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k(cat) value and the k(cat)/K(m) value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.     

A barley polyamine oxidase isoform with distinct structural features and subcellular localization.

Two cDNAs encoding polyamine oxidase (PAO) isoforms (BPAO1 and BPAO2) and the corresponding gene copies were isolated from barley cultivar Aura. Gene organization is not conserved between these two nonallelic coding sequences. Both precursor proteins include a cleavable N-terminal leader of 25 amino acids. N-terminal sequencing of PAO purified from barley seedlings reveals a unique amino-acid sequence corresponding to the BPAO2 N-terminus as predicted from the corresponding cDNA. BPAO2 has been purified, characterized and compared to maize PAO (MPAO), the best characterized member of this enzyme class. The two proteins show different pH optima for catalytic activity, Km and Vmax values with spermidine and spermine as substrates. Molecular modelling of BPAO2 reveals the same global fold as in MPAO. However, substitution of the active site residue Phe403 by a tyrosine, provides a rationale for the different catalytic properties of the two enzymes. In barley leaves PAO-specific activity is higher in isolated mesophyll protoplasts than in the extracellular fluids, whereas in maize the reverse is true. The C-terminus of BPAO2 shows homology with the endoplasmic reticulum retention signal that might be responsible for the subcellular localization observed. We conclude that BPAO2 is a symplastic PAO in barley mesophyll cells. Production of BPAO2 mRNA and the corresponding protein is induced by light, and has a different pattern of accumulation in leaves and coleoptiles.     

Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase

Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N(5) atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pK(a) of approximately 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.     

Metabolism of acetylpolyamines by monoamine oxidase,diamine oxidase and polyamine oxidase

N¹-Monoacetylspermine, N¹,N¹²-diacetylspermine and N¹-monoacetylspermidine were found to be good substrates for rat liver polyamine oxidase, but not for rat liver mitochondrial monoamine oxidase. N⁸-Monoacetylspermidine, monoacetylcadaverine, monoacetylputrescine and monoacetyl-1,3-diaminopropane were oxidized by the monoamine oxidase when the substrate concentration was 10.0 mM, but not by the polyamine oxidase. All the acetylpolyamines except N¹,N¹²-diacetylspermine were also oxidized by hog kidney diamine oxidase although their affinities for the oxidase appeared low. The present data suggest that acetylpolyamines are not easily metabolized in vivo by either monoamine oxidase or diamine oxidase in mammalian tissues although N¹-monoacetylspermine, N¹,N¹²-diacetylspermine and N¹-monoacetylspermidine are attacked by polyamine oxidase.     

The synergistic role of Pu.1 and Fms in zebrafish osteoclast-reducing osteopetrosis and possible therapeutic strategies

Osteoclasts are bone resorption cells of myeloid origin. Osteoclast defects can lead to osteopetrosis, a genetic disorder characterized by bone sclerosis for which there is no effective drug treatment. It is known that Pu.1 and Fms are key regulators in myelopoiesis, and their defects in mice can lead to reduced osteoclast numbers and consequent osteopetrosis. Yet how Pu.1 and Fms genetically interact in the development of osteoclasts and the pathogenesis of osteopetrosis is still unclear. Here, we characterized pu.1^G242D;fms^j4e1 double-deficient zebrafish, which exhibited a greater deficiency of functional osteoclasts and displayed more severe osteopetrotic symptoms than the pu.1^G242D or fms^j4e1 single mutants, suggesting a synergistic function of Pu.1 and Fms in the regulation of osteoclast development. We further demonstrated that Pu.1 plays a dominant role in osteoclastogenesis, whereas Fms plays a dominant role in osteoclast maturation. Importantly, treatment with the drug retinoic acid significantly relieved the different degrees of osteopetrosis symptoms in these models by increasing the number of functional osteoclasts. Thus, we report the development of valuable animal models of osteopetrosis, and our results shed light on drug development for antiosteopetrosis therapy.     

Involvement of polyamine oxidase in wound healing

Hydrogen peroxide (H(2)O(2)) is involved in plant defense responses that follow mechanical damage, such as those that occur during herbivore or insect attacks, as well as pathogen attack. H(2)O(2) accumulation is induced during wound healing processes as well as by treatment with the wound signal jasmonic acid. Plant polyamine oxidases (PAOs) are H(2)O(2) producing enzymes supposedly involved in cell wall differentiation processes and defense responses. Maize (Zea mays) PAO (ZmPAO) is a developmentally regulated flavoprotein abundant in primary and secondary cell walls of several tissues. In this study, we investigated the effect of wounding on ZmPAO gene expression in the outer tissues of the maize mesocotyl and provide evidence that ZmPAO enzyme activity, protein, and mRNA levels increased in response to wounding as well as jasmonic acid treatment. Histochemically detected ZmPAO activity especially intensified in the epidermis and in the wound periderm, suggesting a tissue-specific involvement of ZmPAO in wound healing. The role played by ZmPAO-derived H(2)O(2) production in peroxidase-mediated wall stiffening events was further investigated by exploiting the in vivo use of N-prenylagmatine (G3), a selective and powerful ZmPAO inhibitor, representing a reliable diagnostic tool in discriminating ZmPAO-mediated H(2)O(2) production from that generated by peroxidase, oxalate oxidase, or by NADPH oxidase activity. Here, we demonstrate that G3 inhibits wound-induced H(2)O(2) production and strongly reduces lignin and suberin polyphenolic domain deposition along the wound, while it is ineffective in inhibiting the deposition of suberin aliphatic domain. Moreover, ZmPAO ectopic expression in the cell wall of transgenic tobacco (Nicotiana tabacum) plants strongly enhanced lignosuberization along the wound periderm, providing evidence for a causal relationship between PAO and peroxidase-mediated events during wound healing.     

Occurrence of polyamine oxidase in tissues of the catfish

1. Polyamine oxidase (PAO) activity was found in the brain, intestine, kidney and liver of the siluroid catfish using N1-acetylspermine as substrate. It was highest in the intestine and lowest in the brain. 2. Substrate specificity of the enzyme was tested in the intestine and liver and the highest activity was found with N1-acetylspermine, followed by N1-acetylspermidine and N1,N12-diacetylspermine. 3. The apparent Km values for N1-acetylspermine were 19.6 and 46.9 microM for the intestine and liver, respectively. 4. These results suggest the presence of a system of polyamine reutilization after their acetylation in fishes.     

In vitro and in vivo inhibition of plant polyamine oxidase activity by polyamine analogues

Polyamine oxidase from Avena sativa L. cv. Cristal seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme had a high affinity for spermidine and spermine (K_m ∼ 5.5 and 1.2 μM, respectively), and also oxidized norspermidine (K_m ∼ 64.0 μM). Natural and synthetic diamines, cyclohexylamine, the putrescine analogue 1-aminooxy-3-aminopropane, and several polyamine analogues had inhibitory effects on polyamine oxidase activity and none were substrates. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. By contrast, 1-aminooxy-3-aminopropane showed mixed inhibition kinetics and a K_i value of 0.113 mM. In addition, in vitro enzymatic activity assays showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene], the tetramine 1,14-bis-[ethylamino]-5,10-diazatetradecane, and the pentamine 1,19-bis-[ethylamino]-5,10,15-triazanonadecane, displayed potent competitive inhibitory activities against polyamine oxidase with K_i values of 5.8, 110.0 and 7.6 nM, respectively, where cyclohexylamine was a weak competitive inhibitor with a K_i value of 0.5 mM. These analogues did not inhibit mycelial growth of the fungus Sclerotinia sclerotiorum (Lib.) De Bary and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with concentrations similar to those used for polyamine analogues, guazatine (a well-known fungicide and at the same time, a polyamine oxidase inhibitor) inhibited (∼85%) S. sclerotiorum mycelial growth on Czapek-Dox medium.Finally, the analogue 1,19-bis-ethylamino-5,10,15-triazanonadecane inhibited polyamine oxidase activity observed in segments of maize leaves in vivo. The results obtained provide insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.     

Specific inhibition of polyamine oxidase in vivo is a method for the elucidation of its physiological role

N1-Methyl-N2-(2,3-butadienyl)-1,4-butanediamine (MDL 72521) and N1,N2-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) are specific, potent, enzyme-activated, irreversible inhibitors of polyamine oxidase in vitro. These compounds are also capable of completely inhibiting polyamine oxidase in mouse tissues at intraperitoneal doses greater than 20 mg/kg. Enzyme activity reappears in the various organs within 2-3 days to 50% of the control values. Irreversible inhibition of polyamine oxidase in mice led to decreased putrescine (30-40%) and spermidine (10-20%) levels in liver and some other organs. At the same time N1-acetylspermidine and, to a lesser extent, N1-acetylspermine were accumulating at rates which are assumed to be related to the rates of polyamine degradation. Even after treatment with polyamine oxidase inhibitors over a period of 6 weeks at doses which produced complete inhibition of polyamine oxidase in all organs, including the brain, neither toxic effects nor changes in body weight or behaviour were observed.     

A conceptual model of the polyamine binding site of N1-acetylpolyamine oxidase developed from a study of polyamine derivatives

We used various polyamine derivatives to study the substrate binding site of N ¹-acetylpolyamine oxidase (PAO) that was partially purified from rat liver. The substrate activities of acetylpolyamines indicated the presence of two anionic centers corresponding to the 1,3-diaminopropane (1,3-DAP) structure and a hydrophobic region in addition to the cleavage site of the acetamidopropyl group. Based on the results of the inhibitory activities of 1,3-DAP derivatives, we developed a conceptual model of the polyamine binding site of PAO. We used this model to identify a potent competitive inhibitor, N ¹,N ⁷-dihexyl-1,7-diamino-4-azaheptane, and to develop an affinity column, 1,16-diamino4,13-diazahexadecane–linked Sepharose, which was useful for the purification of PAO.     

Purification and properties of the polyamine oxidase of barley plants

The polyamine oxidase of barley shoots is associated with a particle which sediments in low centrifugal fields. The enzyme was removed from these particles by washing in 0·5 M NaCl and then purified about 24-fold. The purified enzyme oxidized spermine stoicheiometrically to 1,3-diaminopropane and 1-(3-aminopropyl)pyrroline (pH optimum 4·0). Spermidine was oxidized to 1,3-diaminopropane and 1-pyrroline (pH optimum 6·6). At their respective pH optima, spermine is oxidized about 30 times faster than spermidine. Hydrogen peroxide was formed in the course of the polyamine oxidation. The enzyme was not sensitive to several copper chelating reagents but 2-hydroxyethylhydrazine caused 50% inhibition at 5 × 10⁻⁴ M. The enzyme was also present in particles in the roots of barley seedlings and in extracts of the leaves of oats, maize, rye and wheat.     

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N1-(n-octanesulfonyl)spermine and human colon cancer cells

N(1)-(n-octanesulfonyl)spermine (N(1) OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N(1) OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N(1)OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N(1)OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N(1)OSSpm is an example, are suited to preferentially kill tumour cells.     

Mechanistic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: single turnover reaction

The final step in the biosynthesis of the plant hormone ethylene is catalyzed by the non-heme iron-containing enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO). ACC is oxidized at the expense of O(2) to yield ethylene, HCN, CO(2), and two waters. Continuous turnover of ACCO requires the presence of ascorbate and HCO(3)(-) (or an alternative form), but the roles played by these reagents, the order of substrate addition, and the mechanism of oxygen activation are controversial. Here these issues are addressed by development of the first functional single turnover system for ACCO. It is shown that 0.35 mol ethylene/mol Fe(II)ACCO is produced when the enzyme is combined with ACC and O(2) in the presence of HCO(3)(-) but in the absence of ascorbate. Thus, ascorbate is not required for O(2) activation or product formation. Little product is observed in the absence of HCO(3)(-), demonstrating the essential role of this reagent. By monitoring the EPR spectrum of the sample during single turnover, it is shown that the active site Fe(II) oxidizes to Fe(III) during the single turnover. This suggests that the electrons needed for catalysis can be derived from a fraction of the initial Fe(II)ACCO instead of ascorbate. Addition of ascorbate at 10% of its K(m) value significantly accelerates both iron oxidation and ethylene formation, suggesting a novel high-affinity effector role for this reagent. This role can be partially mimicked by a non-redox-active ascorbate analog. A mechanism is proposed that begins with ACC and O(2) binding, iron oxidation, and one-electron reduction to form a peroxy intermediate. Breakdown of this intermediate, perhaps by HCO(3)(-)-mediated proton transfer, is proposed to yield a high-valent iron species, which is the true oxidizing reagent for the bound ACC.     

Molecular evolution of the polyamine oxidase gene family in Metazoa

ABSTRACT: BACKGROUND: Polyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived. RESULTS: We analysed 36 SMO, 26 APAO and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all the SMOs and APAOs from vertebrates. The two vertebrate monophyletic clades clustered strictly mirroring the organismal phylogeny of fishes, amphibians, reptiles, birds, and mammals. Evidences from comparative genomic analysis, structural evolution and functional divergence in a phylogenetic framework across Metazoa suggested an evolutionary scenario where the ancestor PAO coding sequence, present in invertebrates as an orthologous gene, has been duplicated in the vertebrate branch to originate the paralogous SMO and APAO genes. A further genome evolution event concerns the SMO gene of placental, but not marsupial and monotremate, mammals which increased its functional variation following an alternative splicing (AS) mechanism. CONCLUSIONS: In this study the explicit integration in a phylogenomic framework of phylogenetic tree construction, structure prediction, and biochemical function data/prediction, allowed inferring the molecular evolutionary history of the PAO gene family and to disambiguate paralogous genes related by duplication event (SMO and APAO) and orthologous genes related by speciation events (PAOs, SMOs/APAOs). Further, while in vertebrates experimental data corroborate SMO and APAO molecular function predictions, in invertebrates the finding of a supported phylogenetic clusters of insect PAOs and the co-occurrence of two PAO variants in the amphioxus urgently claim the need for future structure-function studies.     

Properties of purified recombinant human polyamine oxidase,PAOh1/SMO

The discovery of an inducible oxidase whose apparent substrate preference is spermine indicates that polyamine catabolism is more complex than that originally proposed. To facilitate the study of this enzyme, the purification and characterization of the recombinant human PAOh1/SMO polyamine oxidase are reported. Purified PAOh1/SMO oxidizes both spermine (K(m)=1.6 microM) and N(1)-acetylspermine (K(m)=51 microM), but does not oxidize spermidine. The purified human enzyme also does not oxidize eight representative antitumor polyamine analogues; however, specific oligamine analogues were found to be potent inhibitors of the oxidation of spermine by PAOh1/SMO. The results of these studies are consistent with the hypothesis that PAOh1/SMO represents a new addition to the polyamine metabolic pathway that may represent a new target for antineoplastic drug development.     

Further properties of the polyamine oxidase of barley leaves

Polyamine analogues have been studied as potential inhibitors or substrates of barley leaf polyamine oxidase. NH₂(CH₂)₃NH(CH₂)₁₀NH₂ was particularly effective as an inhibitor of spermine oxidation at pH 4·5 (K_i = 5 × 10^?6 M). Methylglyoxal-bis(guanylhydrazone) inhibited spermine oxidation only slightly (K_i = 10^?4 M). Activity with the polyamine analogues as substrates was generally 10% or less of the activity with spermine. The K_m for oxygen was 3 × 10^?4 M. The K_m for spermine oxidation was independent of oxygen concentration. Using the N-methyl-2-benzothiazolone hydrazine reagent, 1-(3-aminopropyl)pyrroline was shown to be formed stoichiometrically by the enzyme on oxidation of spermine. The enzyme will not function as a dehydrogenase in the presence of oxygen with either potassium ferricyanide or dichlorophenolindophenol as electron acceptors. Activity in the leaves increased with age, up to 4 weeks. In the leaves of 11-week-old plants activity was lower than in leaves of 1-week-old plants. The enzyme was mainly associated with an easily-sedimented particulate fraction, and relatively small proportions were found in the cell wall or soluble fractions.