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.     

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.     

An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha.

The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis. The D319E mutant catalyzes the oxidation of methylamine and phenethylamine, but not that of benzylamine. kcat/Km for methylamine is found to be 80-fold reduced compared to that of the wild type. Viscosogen and substrate and solvent deuteration have no effect on this parameter for D319E, which is suggestive of limitation of kcat/Km by a conformational change. This conformational change is proposed to be the movement of the cofactor into a productive orientation upon the binding of substrate. In the absence of substrate, a flipped cofactor orientation is likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen. kcat for D319E methylamine oxidase is reduced 200-fold compared to that of the wild type and is unaffected by substrate deuteration, but displays a substantial solvent isotope effect. A 428 nm absorbance is evident under conditions of saturating methylamine and oxygen with D319E. The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity. Resonance Raman spectroscopy indicates the formation of a covalent ammonia adduct and identifies it as the deprotonated iminoquinone. In contrast, when the D319E mutant is reacted with ammonia, it gives predominantly a 340-350 nm species. This absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct formation. Resonance Raman spectroscopic examination of the steady state species of D319E methylamine oxidation, in combination with the kinetic data, indicates that the 428 nm species is the deprotonated iminoquinone produced upon reoxidation of the reduced cofactor. A model is proposed in which a central role of the active site base is to position the free cofactor and several enzyme intermediates for optimal activity.     

Structural and catalytic properties of copper in lysyl oxidase

The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.     

EPR studies of spin-labeled bovine plasma amine oxidase: the nature of the substrate-binding site.

The carbonyl cofactor of bovine plasma amine oxidase (EC 1.4.3.6), recently shown to be 6-hydroxydopa (also known as topa), has been spin labeled to the extent of one label per enzyme dimer molecule, using 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) and 4-hydrazino-TEMPO followed by reduction with borohydride. By studying the EPR spectra of the labeled enzyme, it has been deduced that there is no magnetic interaction between the copper and the spin label, and that the spin label is at least 1.3 nm distant from the copper(II) ion in the resting enzyme. The bound label is strongly immobilized, is in a sterically constricted environment, and is not accessible to small anions. Removal of the copper does not alter the EPR spectrum of the label. The results are similar to results for porcine plasma amine oxidase, and show that the copper is not close to, and does not directly interact with, the topa-bound substrate.     

Methylamine dehydrogenase of Pseudomonase sp. J Isolation and properties of the subunits.

Two kinds of subunits, light subunit (Mr =1300) and heavy subunit (Mr=40 000), were isolated from a methylamine dehydrogenase (Mr=105 000) of Pseudomonas sp. J. The isolation of the subunits was carried out by gel chromatography after the enzyme had been treated with 3M guanidine-HCl. Coexistence of both of the subunit exhibited an absorption maximum only at 278 nm but in addition to the peak at 278 nm. The results indicate that the prosthetic group, assumed to be a derivative of pyridoxal, was bound to the light subunit. The spectral changes of the light subunit were observed by addition of methylamine. Various physical and biochemical parameters of the subunits are reported.     

Roles of the two copper ions in bovine serum amine oxidase

With a view to obtaining information on the roles of the two copper ions in bovine serum amine oxidase (BSAO), spectroscopic and magnetic studies on several BSAO derivatives have been carried out. Cu-depleted BSAO (Cu-depBSAO) exhibits no enzyme activity and only a low absorption intensity at ca. 475 nm, which is the characteristic absorption maximum of the chromophore in BSAO. The binding of 1 mol of Cu to 1 mol of Cu-depBSAO slightly but definitely increases the enzyme activity and the absorptivity, although they are much lower than those of native BSAO. The incorporation of 2 mol of Cu into Cu-depBSAO gives rise to a similar high activity and absorptivity as those of the native enzyme. Electron paramagnetic resonance (EPR) spectra of the BSAO derivatives reveal that two copper ions in the enzyme molecule are environmentally identical. Titrations of BSAO, Cu-depBSAO, and Cu-half-depleted BSAO (Cu-half-depBSAO), containing 1 mol of copper per mole of protein, with phenylhydrazine (an inhibitor of BSAO) indicate that only 1 mol of phenylhydrazine reacts with 1 mol of the enzyme. In other words the enzyme possesses only one chromophore or one active site, though the molecule is composed of two electrophoretically identical subunits. The binding constants between phenylhydrazine and BSAO, Cu-depBSAO, or Cu-half-depBSAO were estimated to be 5 X 10(6), 5 X 10(4), and 1 X 10(5) M-1, respectively. The binding of phenylhydrazine to the chromophore is assisted by the presence of two copper ions by a factor of 100.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of blue copper proteins in the oxidation of methylamine by an obligate methylotroph.

Organism 4025, an obligate methylotroph, when grown on methylamine in the presence of a high concentration of copper, contained high concentrations of methylamine dehydrogenase and two blue copper proteins, amicyanin and an azurin-type protein; these were purified to homogeneity and characterized. The methylamine dehydrogenase is a basic protein (pI 8.8) and consists of light and heavy subunits (Mr 14100 and 43000; total Mr 112000). This dehydrogenase differed slightly from other methylamine dehydrogenases in its absorption spectrum and in its lack of thermal stability. Amicyanin, the more abundant blue copper protein, had an Mr of 11500, a midpoint redox potential of 294mV at pH 7.0, and a much lower isoelectric point (pI5.3) than other amicyanins. Its absorption maximum was 620 nm (7-24 nm higher than those of other amicyanins); its absorption coefficient (at 620 nm) was 3.8 mM-1 X cm-1. The 'azurin' (6% of the blue copper protein) had an Mr of 12500, a midpoint redox potential of 323 mV and a high isoelectric point (pI 9.4). Its absorption maximum was 620 nm, the absorption coefficient (16 mM-1 X cm-1) at this wavelength being considerably greater than that of any blue copper protein described previously. The partially-purified soluble cytochromes cH and cL were similar to those of other methylotrophs. The interactions of the purified redox proteins were investigated in order to elucidate their role in methylamine oxidation. Methylamine dehydrogenase was able to donate electrons only to amicyanin, the rate of reaction being 2.04 mmol/min per mumol of methylamine dehydrogenase; this is sufficient to account for the rate of respiration in whole bacteria. The blue copper proteins were able to react rapidly with each other and with both the soluble cytochromes c.     

Ascorbate oxidase from Cucurbita pepo medullosa. New method of purification and reinvestigation of properties.

1. Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low-molecular-weight copper protein containing one type-1 copper/20000 Mr could be separated during the purification of the oxidase. The six-step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 +/- 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 +/- 50 units/mg protein or 1088 +/- 15 units/microgram copper. 2. The pure enzyme is characterized by the following optical purity indices: A280/A610 = 25 +/- 0.5, A330/A610 = 0.65 +/- 0.05 and A610/A500 = 7.0 +/- 0.25. The molar absorption coeffient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts of 9700 M-1 cm-1 . 3. Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type-1 (blue) copper gz = 2.227, gy = 2.058, gx = 2.036; Az = 5.0 mT, Ay = Ax = 0.5 mT, for the type-2 (non-blue) copper g parallel to = 2.242, g perpendicular = 2.053; A parallel to = 19.0 mT, A perpendicular 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type-1 class, and one to the type-2 class, as demonstrated by computer simulations of the EPR spectra. 4. To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L-ascorbate or reductase must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (lambda max ex = 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type-3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310 nm (delta epsilon = 1000 +/- 50 M-1 cm-1). Corresponding changes in EPR and fluorescence spectra have not been detected.     

Confirmation of the presence of a Cu(II)/topa quinone active site in the amine oxidase from fenugreek seedlings

Amine oxidase from etiolated seedlings of fenugreek (Trigonellafoenum—graecum) has been isolated by a purification procedureinvolving three chromato—graphic steps. The homogeneousenzyme is of pink colour with a visible absorption maximum at500 nm. The dimeric enzyme (2 75 kDa) is a slightly acidicprotein (pl 6.8) containing 8% neutral sugars. N—ter—minalamino acid sequence of the enzyme shows a high degree of similarityto other plant and microbial copper—containing amine oxidases.The best substrates of the enzyme are aliphatic diamines andsome polyamines, whereas inhibitors are substrate analogues,copper complexing agents, some alkaloids and several other compounds.Spectrophotometric titra—tions with phenylhydrazines demonstratedone reactive carbonyl group per subunit of the enzyme and redox—cyclicquinone staining after native electrophor—esis indicatedthe presence of a quinone cofactor. Differential pulse polarographyshowed the existence of a copper/quinone—containing activesite. The resonance Raman spectroscopy and the pH—dependentshift of the absorption spectrum of the enzyme p—nitrophenylhydrazoneconfirm unambiguously the identity of the cofactor with topaquinone. EPR spectra of the enzyme are in accordance with thoseof tetragonal cupric complexes as known for other copper—containingamine oxidases. Besides the copper, Mn(II)ions were detectedthat partially occupy another metal site in the enzyme, buttheir catalytical importance is unlikely. Key words: Fenugreek, Trigonella foenum—graceum, amine oxidase, topa quinone     

Microbial oxidation of amines. Spectral and kinetic properties of the primary amine dehydrogenase of Pseudomonas AM 1

1. An improved procedure is reported for purification of the amine dehydrogenase from methylamine-grown Pseudomonas AM1 which yielded a product homogeneous by sedimentation and disc-electrophoretic analysis, with molecular weight of 133000. 2. The purified enzyme had absorption maxima at 280 and 430nm. On aging, a third peak appeared at 325nm, and the 430nm peak decreased in intensity. This spectrum was independent of pH. 3. Addition of 2.5mm-semicarbazide, phenylhydrazine, hydrazine or hydroxylamine produced modified spectra with maxima respectively at 400, 440, 395 and 425nm. 4. Aerobic addition of methylamine resulted in a bleaching of the 430nm peak and the appearance of a new one at 325nm. This spectral change was retained after removal of the methylamine by dialysis. The original spectrum could be restored on addition of phenazine methosulphate. 5. Addition of borohydride partially inactivated the enzyme and produced spectral changes similar to those observed with methylamine. Pre-treatment with methylamine prevented the inactivation by borohydride. The degree of inactivation could be increased by alternate phenazine methosulphate and borohydride treatments. 6. The addition of methylamine or borohydride each caused shifts in the fluorescence emission maximum from 348 to 380nm. 7. Lineweaver-Burk plots of reciprocal activity against reciprocal concentration of either of the substrates n-butylamine or phenazine methosulphate were consistent with a mechanism that involves interconversion of two free forms of the enzyme by the two substrates. 8. The enzyme, although spectrally modified, was not inactivated by dialysis against diethyldithiocarbamate, and contained about 0.27 g-atom of copper/mol, with small traces of cobalt, iron and zinc. 9. Conventional methods of resolution did not release the prosthetic group. Heat denaturation after treatment of the enzyme with methylamine liberated a yellow chromophore which did not reactivate resolved aspartate aminotransferase, and whose spectral, electrophoretic and fluorescence properties did not agree with any recognizable pyridoxal derivatives. 10. Despite the inconclusive results with the isolated chromophore, the observations on the enzyme suggest that it may contain a pyridoxal derivative bound as a Schiff's base which is converted into the pyridoxamine form on aerobic treatment with methylamine and reconverted into the pyridoxal form with phenazine methosulphate. 11. The copper detected is probably not involved in the enzyme mechanism, since most copper-chelating agents are not inhibitory, and since the enzyme does not react with oxygen.     

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.     

Multiple structures and functions of cytochrome oxidase

X-ray absorption studies have been used to investigate the structure of the four redox centers (2Fe, 2Cu) of the terminal enzyme in the respiratory chain, cytochrome c oxidase in the resting oxidized form as well as in the functional intermediates that are freeze-trapped. Methods of x-ray fluorescence detection for these low-concentration samples together with low-temperature cryostats and simultaneous optical monitoring were developed to ensure good signal-to-noise data and sample integrity. The resting oxidized form contains a sulfur bridge between the copper and iron of the active site which are separated by approximately 3.8 A. This separation of the active site metal atoms was uniquely identified by comparison of both the iron and copper EXAFS data and iron EXAFS of the copper-depleted enzyme. In the reduced state, the CO or O2 is bound to the active site iron having a structure identical to CO or oxy hemoglobin while the sulfur remains with the active site copper. Little change in structure is observed for the other iron and copper. It is the sulfur bridged active site form that is isolated by the Yonetani and Caughy methods with greater than or equal to 85% homogeneity but not the Hartzell-Beinert or similar methods. Another form observed in the redox cycle is also fully oxidized but lacks the sulfur bridged active site with the iron of the active site having a structure identical to that of the peroxidases. This form exhibits peroxidase as well as oxidase activity, and a stable intermediate is formed with hydrogen and ethylhydrogen peroxide in which the iron of the active site is structurally similar to that of the peroxidase intermediate. The active site copper, however, does not participate in the peroxidatic role and the structures of the other iron and copper are identical to those of the sulfur bridged resting oxidized form. Thus this unique enzyme has peroxidase activity which may serve to safeguard its main oxidase function.     

Inhibition of bovine plasma semicarbazide-sensitive amine oxidase by caffeine

Semicarbazide-sensitive amine oxidase (SSAO) is a copper-containing enzyme that catalyzes the oxidative deamination of endogenous and exogenous primary amines. SSAO exists in mammals both as a plasma-soluble and as a membrane-bound form, and its active site is able to come into contact with numerous xenobiotic, amine-containing compounds. The kinetic studies performed in this work showed that caffeine inhibition of bovine serum amine oxidase was noncompetitive when benzylamine was used as substrate and mixed when the substrate used was methylamine. Since caffeine contains an imidazole ring, it cannot be excluded that it might bind to an inhibitory imidazoline-binding site on SSAO.     

The crystal structure of Pichia pastoris lysyl oxidase

Pichia pastoris lysyl oxidase (PPLO) is unique among the structurally characterized copper amine oxidases in being able to oxidize the side chain of lysine residues in polypeptides. Remarkably, the yeast PPLO is nearly as effective in oxidizing a mammalian tropoelastin substrate as is a true mammalian lysyl oxidase isolated from bovine aorta. Thus, PPLO is functionally related to the copper-containing lysyl oxidases despite the lack of any significant sequence similarity with these enzymes. The structure of PPLO has been determined at 1.65 A resolution. PPLO is a homodimer in which each subunit contains a Type II copper atom and a topaquinone cofactor (TPQ) formed by the posttranslational modification of a tyrosine residue. While PPLO has tertiary and quaternary topologies similar to those found in other quinone-containing copper amine oxidases, its active site is substantially more exposed and accessible. The structural elements that are responsible for the accessibility of the active site are identified and discussed.     

Reductive trapping of substrate to methylamine oxidase from Arthrobacter P1

Methylamine oxidase (EC 1.4.3.6) from Arthrobacter P1 was inactivated by NaCNBH3 in the presence of [14C]benzylamine, leading to the incorporation of 1 mol of radiolabeled substrate/mol of enzyme subunit at complete inactivation. By contrast, no labeling of enzyme was observed using [3H]NaCNBH3 as reductant. These results are analogous to those previously reported for the eukaryotic enzyme, bovine serum plasma amine oxidase [(1987) J. Biol. Chem. 262, 962-965]. The observed pattern of labeling is consistent with the presence of dicarbonyl cofactor at the active site of methylamine oxidase. Further, these studies suggest that our reductive trapping technique, in which the pattern of radiolabeling of an enzyme is compared using C-14 substrate vs tritiated reductant, may serve as a general assay for covalently bound dicarbonyl structures.     

Arthrobacter P 1, a fast growing versatile methylotroph with amine oxidase as a key enzyme in the metabolism of methylated amines

A facultative methylotrophic bacterium was isolated from enrichment cultures containing methylamine as the sole carbon source. It was tentatively identified as an Arthrobacter species. Extracts of cells grown on methylamine or ethylamine contained high levels of amine oxidase (E.C. 1.4.3.) activity. Glucose- or choline-grown cells lacked this enzyme. Oxidation of primary amines by the enzyme resulted in the formation of H₂O₂; as a consequence high levels of catalase were present in methylamine-and ethylamine-grown cells. The significance of catalase in vivo was demonstrated by addition of 20 mM aminotriazole (a catalase inhibitor) to exponentially growing cells. This completely blocked growth on methylamine whereas growth on glucose was hardly affected. Cytochemical studies showed that methylamine-dependent H₂O₂ production mainly occurred on invaginations of the cytoplasmic membrane. Assimilation of formaldehyde which is generated during methylamine oxidation was by the FBP variant of the RuMP cycle of formaldehyde fixation. The absence of NAD-dependent formaldehyde and formate dehydrogenases indicated the operation of a non-linear oxidation sequence for formal-dehyde via hexulose phosphate synthase. Enzyme profiles of the organism grown on various substrates suggested that the synthesis of amine oxidase, catalase and the enzymes of the RuMP cycle is not under coordinate control.     

Preparation of a one-subunit cytochrome oxidase from Paracoccus denitrificans: spectral analysis and enzymatic activity

Cytochrome c oxidase was isolated from Paracoccus denitrificans as a two-subunit enzyme. Chymotrypsin-catalyzed proteolysis reduced the molecular weight of each subunit by about 8000. The spectral properties of this preparation, as well as its Km for cytochrome c(1.7 muM), remained unchanged with respect to the native enzyme. Vmax was reduced by about 55% when assayed in Triton X-100 or in Triton X-100 supplemented with asolectin. Following further proteolysis by Staphylococcus aureus V8 protease, subunit I remained unchanged as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas subunit II was split into small peptides. These were removed by ion-exchange high-performance liquid chromatography. The one-subunit enzyme had an apparent molecular weight of 43,000. The reduction of molecular weight was also confirmed by the diminution of the ultraviolet/Soret absorption ratio. This value was 1.8-2.1 for the native enzyme and 1.3-1.5 for the one-subunit enzyme. The spectral properties (including the spectrum CO reduced minus reduced) were not modified by the proteolytic treatment, indicating that cytochromes a and a3 were present in equal amounts. The lack of spectral alteration and the known close association of the copper B atom with cytochrome a3 suggest that copper B is also contained within the one-subunit enzyme. The Km of the one-subunit oxidase was similar to that of the two-subunit enzyme; Vmax was decreased by about 50%. The activity of the one-subunit oxidase had a salt-dependent maximum at 30 mM KCl, almost identical with that of the undigested enzyme, and was inhibited by micromolar concentrations of KCN.     

Inhibition of lentil copper/TPQ amine oxidase by the mechanism-based inhibitor derived from tyramine

Copper amine oxidase from lentil (Lens esculenta) seedlings was shown to catalyze the oxidative deamination of tyramine and three similar aromatic monoamines, benzylamine, phenylethylamine and 4-methoxyphenylethylamine. Tyramine, an important plant intermediate, was found to be both a substrate and an irreversible inhibitor of the enzyme whereas the other amines were not inhibitory. In the course of tyramine oxidation the enzyme gradually became inactivated with the concomitant appearance of a new absorption at 560 nm due to the formation of a stable adduct. Inactivation took place only in the presence of oxygen and was probably due to the reaction of the enzyme with the oxidation product of tyramine, p-hydroxyphenylacetaldehyde. The kinetic data obtained in this study indicate that tyramine represents a new interesting type of physiological mechanism-based inhibitor for plant copper amine oxidases.     

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.