Effect of metal substitution in copper amine oxidase from lentil seedlings

 The reaction with substrates and carbonyl reagents of native lentil Cu-amine oxidase and its modified forms, i.e. Cu-fully-depleted, Cu-half-reconstituted, Cu-fully-reconstituted, Co-substituted, Ni-substituted and Zn-substituted, has been studied. Upon removal of only one of the two Cu ions, the enzyme loses 50% of its enzymatic activity. Using several substrates, Co-substituted lentil amine oxidase is shown to be active but the k _c value is different from that of native or Cu-fully-reconstituted enzyme, while K _m is similar. On the other hand, the Ni- and Zn-substituted forms are catalytically inactive. Enzymatic activity measurements and optical spectroscopy show that only in the Co-substituted enzyme is the organic cofactor 6-hydroxydopa quinone reactive and the enzyme catalytically competent, although less efficient. The Co-substituted amine oxidase does not form the semiquinone radical as an intermediate of the catalytic reaction. While devoid or reduced of catalytic activity, all the enzyme preparations are still able to oxidise two moles of substrate and to release two moles of aldehyde per mole of dimeric enzyme. The results obtained show that although Co-substituted amine oxidase is catalytically competent, copper is essential for the catalytic mechanism. Received: 5 March 1999 / Accepted: 22 July 1999     

Oxidation of benzylamine Br-derivatives by lentil seedling copper-amine oxidase

ABSTRACT

Copper amine oxidase was shown to be able to catalyse the oxidative deamination of 2-, 3- and 4-Br-derivatives of benzylamine to the corresponding aldehydes, that all absorb at 250 nm. This change in the absorption spectrum made it possible to follow the enzyme reaction. 2-Br-benzylamine, 3-Br-benzylamine, and 4-Br-benzylamine showed K_m values similar to benzylamine, but 3-Br-benzylamine showed a slower k_c, which allows it to be a catalytically more efficient substrate. Under anaerobic conditions the native enzyme oxidised 1 equivalent of all Br-derivatives and released 1 equivalent of aldehyde per enzyme subunit. These findings demonstrate that, in anaerobic conditions, the enzyme can oxidise substrates with a single incomplete turnover. The possible involvement of the cofactor 6-hydroxydopa quinone and of a negatively charged residue in the oxidation of Br-benzylamines is discussed.     

Reactivity study on microperoxidase-8

The catalytic activity of the microperoxidase-8/H₂O₂ system toward tyramine and 3-(4-hydroxyphenyl)propionic acid has been determined in acetate buffer, pH 5.0. Operating with a strong excess of hydrogen peroxide, the rate-determining step of the reaction was substrate oxidation. Owing to the fast microperoxidase-8 degradation, only the very initial phase of the reactions were analyzed. The reaction rates follow a substrate saturation behavior, with turnover numbers [k_cat=26±1 s^–1 for 3-(4-hydroxyphenyl)propionic acid and k_cat=22±1 s^–1 for tyramine] that were similar for the two substrates. In contrast, the K_M values indicated a reduced affinity for the catalyst active species by the positively charged phenol, probably due to repulsive interaction with the protonated N-terminal microperoxidase-8 amino group. The reactivity of the catalyst active species was studied upon incubation of microperoxidase-8 with a small excess hydrogen peroxide, followed by reaction with the phenolic substrates. The kinetic analysis showed that more than two active species are accumulated. The species responsible for the faster reactions was present in solution as a minor fraction. The active intermediate which accumulated in a larger amount (intermediate III) has a reduced substrate oxidation activity. Comparison of this activity with the kinetic constants obtained under turnover experiments shows that intermediate III is not involved in the microperoxidase-8 catalytic cycle. The active species of the catalytic process are intermediates I and II, which in the absence of substrate rapidly convert to intermediate III.Abbreviations ABTS 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) - HPA 3-(4-hydroxyphenyl)propionic acid - HRP horseradish peroxidase - MP-8 microperoxidase-8     

Human enteropeptidase light chain: Bioengineering of recombinants and kinetic investigations of structure and function

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK～ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (k_cat/K_M = 6.83 × 10⁶ M^?1 sec^?1) and DDDDR (k_cat/K_M = 1.89 × 10⁷ M^?1 sec^?1) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge–charge interactions in the extended substrate binding pocket.     

Catechol(amine)s as probes of lactoperoxidase catalytic site structure: spectroscopic and modeling studies

 Binding affinities to lactoperoxidase (LPO) of a homologous series of substituted catechol(amine)s [such as catechol, 4-methylcatechol, 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, 3-(3,4-dihydroxyphenyl)propionic acid; dopamine, noradrenaline, adrenaline;l-3,4-dihydroxyphenylalanine] were studied by UV-visible spectroscopy and docking simulations. Dissociation constant (K _d) values were calculated by direct fitting of the experimental data and fall in a range of 3–95 mM. Thermodynamic parameters are comparable with those reported for the interaction of LPO with p-substituted phenols, suggesting a similar general mode of binding. Furthermore, the relative contributions to binding energy, described by the unimolecular constant K _u, show that interaction between protein and ligands originates from a relatively large number of groups. Docking and molecular dynamics simulations, in agreement with experimental evidence, predict that the substrate is localized into the access channel in the vicinity of heme distal pocket. This channel is characterized by a hydrophobic patch (six Phe residues) and by a charged contribution (two Glu and one His residues). All of the substrates, except caffeic acid, may approach the protein active site. Positively charged Arg372 acts as a gate above the heme distal pocket and seems to address substrate orientation in relation to the side-chain terminal group. Received: 4 June 1998 / Accepted: 1 October 1998     

The hydrolysis of new lipid-like substrates and trilaurin in monolayers catalyzed with the lipase fromPseudomonas fluorescens

A simple method for determining the enzymic hydrolysis parameters of lipid-like substrates and trilaurin assembled in monolayers at the water-air interface was suggested. At a surface pressure of 10 mN/m, the initial rates of lipolysis were found to be proportional to the decrease in area of the substrate monolayer caused by the enzymic hydrolysis in a single-compartment Langmuir balance. The kinetic parameters for the hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acetylated in position 2 with an amino acid (phenylalanine, leucine, or valine) catalyzed with lipase fromPseudomonas fluorescens were determined. Unlike models of enzymic hydrolysis that neglect the thickness of the substrate monolayer, our method allows the determination of kinetic parameters in standard dimensions. The values ofk _cat for the synthetic pseudoglycerides were found to be significantly higher than that for trilaurin, while the values ofK _m(app) were close. This may be due to the presence of positively charged primary amino groups in the molecules of pseudoglycerides.     

A thermostable triple mutant of pyranose 2-oxidase from Trametes multicolor with improved properties for biotechnological applications

In order to increase the thermal stability and the catalytic properties of pyranose oxidase (P2Ox) from Trametes multicolor toward its poor substrate D-galactose and the alternative electron acceptor 1,4-benzoquinone (1,4-BQ), we designed the triple-mutant T169G/E542K/V546C. Whereas the wild-type enzyme clearly favors D-glucose as its substrate over D-galactose [substrate selectivity (k_cat/K_M)_Glc/(k_cat/K_M)_Gal = 172], the variant oxidizes both sugars equally well [(k_cat/K_M)_Glc/(k_cat/K_M)_Gal = 0.69], which is of interest for food biotechnology. Furthermore, the variant showed lower K_M values and approximately ten-fold higher k_cat values for 1,4-BQ when D-galactose was used as the saturating sugar substrate, which makes this enzyme particularly attractive for use in biofuel cells and enzyme-based biosensors. In addition to the altered substrate specificity and reactivity, this mutant also shows significantly improved thermal stability. The half life time at 60°C was approximately 10 h, compared to 7.6 min for the wild-type enzyme. We performed successfully small-scale bioreactor pilot conversion experiments of D -glucose/D -galactose mixtures at both 30 and 50°C, showing the usefulness of this P2Ox variant in biocatalysis as well as the enhanced thermal stability of the enzyme. Moreover, we determined the crystal structure of the mutant in its unligated form at 1.55 Å resolution. Modeling D-galactose in position for oxidation at C2 into the mutant active site shows that substituting Thr for Gly at position 169 favorably accommodates the axial C4 hydroxyl group that would otherwise clash with Thr169 in the wild-type.     

A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM⁻¹ cm⁻¹) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The K _M value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The K _M of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (k _obs1 = 4.7 s⁻¹, k _obs2 = 1.9 s⁻¹) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (k _obs3 = 6.10 × 10⁻² s⁻¹, k _obs4 = 2.18 × 10⁻² s⁻¹). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.     

A novel enzyme with spermine oxidase properties in bovine liver mitochondria: Identification and kinetic characterization

The uptake of spermine into mammalian mitochondria indicated the need to identify its catabolic pathway in these organelles. Bovine liver mitochondria were therefore purified and their capacity for natural polyamine uptake was verified. A kinetic approach was then used to determine the presence of an MDL 72527-sensitive enzyme with spermine oxidase activity in the matrix of bovine liver mitochondria. Western blot analysis of mitochondrial fractions and immunogold electron microscopy observations of purified mitochondria unequivocally confirmed the presence of a protein recognized by anti-spermine oxidase antibodies in the mitochondrial matrix. Preliminary kinetic characterization showed that spermine is the preferred substrate of this enzyme; lower activity was detected with spermidine and acetylated polyamines. Catalytic efficiency comparable to that of spermine was also found for 1-aminododecane. The considerable effect of ionic strength on the V_max/K_M ratio suggested the presence of more than one negatively charged zone inside the active site cavity of this mitochondrial enzyme, which is probably involved in the docking of positively charged substrates. These findings indicate that the bovine liver mitochondrial matrix contains an enzyme belonging to the spermine oxidase class. Because H₂O₂ is generated by spermine oxidase activity, the possible involvement of the latter as an important signaling transducer under both physiological and pathological conditions should be considered.     

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74‐compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k_cat/K_m values >10,000 M^?1 s^?1: Q92EH0_LISIN of Listeria innocua serovar 6a against ADP‐ribose, Q5LBB1_BACFN of Bacillus fragilis against 5‐Me‐CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8‐oxo‐dATP and 3'‐dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty‐two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k_cat/K_m values exhibited against these canonical substrates are well under 10⁵ M^?1 s^?1. By contrast, several Nudix enzymes show much larger k_cat/K_m values (in the range of 10⁵ to >10⁷ M^?1 s^?1) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810–1822. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Summary Enteropeptidase (enterokinase EC 3.4.21.9), catalyzing trypsinogen activation, exhibits unique properties for high efficiency hydrolysis of the polypeptide chain after the N-terminal tetraaspartyl-lysyl sequence. This makes it a convenient tool for the processing of fusion proteins containing this sequence. We found the enteropeptidase-catalysing degradation of some bioactive peptides: cattle hemoglobin beta-chain fragments Hb (2–8) (LTAEEKA) and Hb (1–9) (MLTAEEKAA), human angiotensin II (DRVYIHPF) (AT). Model peptides with truncated linker WDDRG and WDDKG also were shown to be susceptible to enteropeptidase action. Kinetic parameters of enteropeptidase hydrolysis for these substrates were determined.K _m values for all substrates with truncated linker (≈10⁻³ M) are an order of magnitude higher than corresponding values for typical enteropeptidase artificial peptide or fusion protein substrates with full enteropeptidase linker-DDDDK-(K _m ≈10⁻⁴ M).k _cat values for AT, Hb (2–8), WDDRG and WDDKG are ≈30–40 min⁻¹. But one additional amino acid residue at both N-and C-terminus of Hb (2–8) results in a drastic increase of hydrolysis efficiency:k _cat value for Hb (1–9) is 1510 min⁻¹. Recent study demonstrates the possibility of undesirable cleavage of target peptides or proteins containing the above-mentioned truncated linker sequences; further, the ability of enteropeptidase to hydrolyse specifically several biologically active peptidesin vitro along with its unique natural substrate trypsinogen was demonstrated.     

Microbial oxidation of amines. Distribution, purification and properties of two primary-amine oxidases from the yeast Candida boidinii grown on amines as sole nitrogen source

1. The yeast Candida boidinii was grown on glucose as carbon source with a range of amines and amino acids as nitrogen sources. Cells grown on amines contained elevated activities of catalase. If the amines contained N-methyl groups, formaldehyde dehydrogenase, formate dehydrogenase and S-formylglutathione hydrolase were also elevated in activity compared with cells grown on (NH₄)₂SO₄. 2. Cells grown on all the amines tested, but not those grown on urea or amino acids, contained an oxidase attacking primary amines, which is referred to as methylamine oxidase. In addition, cells grown on some amines contained a second amine oxidase, which is referred to as benzylamine oxidase. 3. Both amine oxidases were purified to near homogeneity. 4. Benzylamine oxidase was considerably more stable at 45 and 50°C than was methylamine oxidase. 5. Both enzymes had a pH optimum in the region of 7.0, and had a considerable number of substrates in common. There were, however, significant differences in the substrate specificity of the two enzymes. The ratio V/K^app._m increased with increasing n-alkyl carbon chain length for benzylamine oxidase, but decreased for methylamine oxidase. 6. Both enzymes showed similar sensitivity to carbonyl-group reagents, copper-chelating agents and other typical `diamine oxidase inhibitors'. 7. The stoicheiometry for the reaction catalysed by each enzyme was established. 8. The kinetics of methylamine oxidase were examined by varying the methylamine and oxygen concentrations in turn. A non-Ping Pong kinetic pattern with intersecting double-reciprocal plots was obtained, giving K_m values of 10μm for O₂ and 198μm for methylamine. The significance of this unusual kinetic behaviour is discussed. Similar experiments were not possible with the benzylamine oxidase, because it seemed to have an even lower K_m for O₂. 9. Both enzymes had similar subunit M_r values of about 80000, but the benzylamine oxidase behaved as if it were usually a dimer, M_r 136000, which under certain conditions aggregated to a tetramer, M_r 288000. Methylamine oxidase was mainly in the form of an octamer, M_r 510000, which gave rise quite readily to dimers of M_r 150000, and on gel filtration behaved as if the M_r was 286000.     

In situ kinetic measurements of d -amino acid oxidase in rat liver with respect to its substrate specificity

Summary d-Amino acid oxidase activity was demonstrated in peroxisomes of rat liver using unfixed cryostat sections and a histochemical technique using cerium ions as capture reagent for hydrogen peroxide and diaminobenzidine, cobalt ions and exogenous hydrogen peroxide to visualize the final reaction product for light microscopical analysis. Cytophotometric analysis of liver sections revealed similar zero-order reaction velocities of d-amino acid oxidase with activity twice as high in periportal areas as in pericentral areas of liver lobuli when using either d-proline or d,l-thiazolidine-2-carboxylic acid as substrates. On the other hand, a 4–5 times higher K _M value was found for d-proline than for d,l-thiazolidine-2-carboxylic acid. The K _M values in periportal and pericentral areas were similar for each substrate. These findings support the suggestion that the physiological substrate for d-amino acid oxidase may be d,l-thiazolidine-2-carboxylic acid, the adduct of cysteamine and glyoxylic acid. d-Amino acid oxidase may play a role in vivo in the production of oxalate which may participate in metabolic control processes as an intracellular messenger molecule.     

Properties and chemical modification of D-amino acid oxidase from Trigonopsis variabilis

The basic properties of purified d-amino acid oxidase from the yeast Trigonopsis variabilis were investigated. The pH optimum of activity was between pH 8.5 and 9.0, and the native molecular masses of holo- and apo-enzyme were determined to be 170 kDa; higher aggregates corresponded to molecular masses of 320 and 570 kDa. The apparent V _max and K _m values for different substrates varied between 3.7 to 185 U/mg and 0.2 to 17.3 mM, respectively. The reaction of d-amino acid oxidase with sulfite was followed by the typical spectral modifications of the FAD resembling the reduced enzyme; a K _d of 30 μM was calculated for the N(5)-adduct. The red anionic flavin radical of the enzyme was stable; benzoate had no influence on the spectral properties. A complete loss of enzyme activity was observed after chemical modification by the histidine-specific reagent diethyl pyrocarbonate. The inactivation showed pseudo-first-order kinetics, with a second-order rate constant of 13.6 M^–1 min^–1 at pH 6.0 and 20°C. The addition of a substrate under anoxic conditions led to a substantial protection from inactivation, which indicates a localization of the modified residues close to the active site. The pK_a of the reacting group was determined to be 7.7, and the rate of inactivation reached a limiting value of 0.031 min^–1. Received: 22 August 1995 / Accepted: 17 October 1995     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

Transport of Charged Dipeptides by the Intestinal H+/Peptide Symporter PepT1 Expressed in Xenopus laevis Oocytes

The cloned intestinal peptide transporter is capable of electrogenic H⁺-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K_0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K_0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5–8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H⁺/H₃O⁺ cotransport may vary depending on pH. Received: 19 August 1996/Revised: 10 October 1996     

Characterization of amine oxidases from Arthrobacter aurescens and application for determination of biogenic amines

Biogenic amines (BAs) that are produced through naturally occurring decarboxylation of amino acids have toxicological effects on humans. Bacterial amine oxidases are useful tools for the rapid quantification of BAs in foods. To develop amine oxidases for the rapid detection of BAs, the genes for amine oxidases from Arthobacter aurescens TC-1, designated AMAO1, AMAO2, and AMAO3, respectively, were cloned and expressed in Escherichia coli. AMAO1 was catalytically inactive to BAs, and AMAO3 showed a narrow substrate spectrum. In contrast, AMAO2 exhibited activity with relative k _cat/K _M values of 100:49.6:7.6 for 2-phenylethylamine, tyramine, and histamine, respectively. AMAO2 also utilized putrescine and spermidine as substrates, with four or five orders of magnitude lower k _cat/K _M values than that of 2-phenylethylamine. AMAO2 and AMAO3 were seriously affected by substrate inhibition. Using BA mixtures (consisting of 2-phenylethylamine, tyramine, and histamine) as samples, the detection range of the enzymatic analysis of BA using AMAO2 was determined to be 2.5–120 μM, and its detection limit was 2.3 μM. Analysis of five commercial cheese products revealed that the BA contents determined by the enzymatic methods showed a good agreement with the sum of three monoamines and histamine by HPLC. Therefore, the enzymatic assay using AMAO2 can be used in quality control of food products through rapid, sensitive, and preliminary estimation of major BAs including the most important TyrN and HisN in foods.     

Kinetic properties of membrane-bound and Triton X-100-solubilized human brain monoamine oxidase

The kinetic properties of membrane-bound and Triton X-100-solubilized human brain mitochondrial type A and B monoamine oxidase were examined. These studies reveal that the K_m values for phenylethylamine and benzylamine, type B monoamine oxidase substrates, were only slightly increased by the solubilization procedure. The K_m value for 5-hydroxytryptamine, a type A monoamine oxidase substrate, was similarly increased by treatment with Triton X-100. The K_m values for oxygen with all three amine substrates were unaffected by solubilization of the oxidase. Similarly, the optimum pH for deamination of substrates for the B isoenzyme was essentially unaltered in the solubilized preparation as compared to the membrane-bound enzyme whereas that for 5-hydroxytryptamine metabolism was decreased from pH 8.5 to approximately 7.75 on solubilization. The energy of activation with all three substrates was altered on solubilization of the oxidases with Triton X-100. The energy of activation for the B monoamine oxidase substrates increased whereas that for 5-hydroxytryptamine decreased. These data support the contention that the lipid environment surrounding the two forms of monoamine oxidase controls, in part, the activity and kinetic properties of the enzymes.     

Non-Productive Binding of Substrates as One of Aspects of Substrate Specificity of Cholinesterases of Different Origin

Taking into account the phenomenon of non-productive binding of substrate, kinetic parameters of hydrolysis of acetylcholine (ACh) and its 13 derivatives with different structures of ammonium group by cholinesterase (ChE) of human erythrocytes, ChE of horse blood serum, and ChE of optic ganglia of the Pacific squid Todarodes pacificus are determined. A dependence is revealed of values of parameters of their enzymatic hydrolysis and parameters of the non-productive binding on the substrate structure and ChE nature. Effects of salts, LiCl, NaCl, KCl, MgCl₂, CaCl₂ and BaCl₂, on various kinetic parameters, including parameters of the non-productive binding of substrate, of enzymatic hydrolysis of iodides of ACh and N-acetoxyethylene-N-ethylpiperidinium under action of horse blood serum ChE are studied. Addition of the salts to the reaction mixture produced different effects on values of the catalytic center activity (a _c) and the Michaelis constant (K _M), depending on the cation nature and the substrate structure. At the same time, values of the a _c/K _M ratio that characterize to a degree the substrate affinity to the enzyme are equal to each other for two substrates differing in structure, regardless of the presence and nature of the studied cations. Parameters of the non-productive binding of N-acetoxyethylene-N-ethylpiperidinium iodide also depended on the salt nature; however, in that case, a question arises as to the correctness of the comparative analysis, when at determinations of the parameters the non-productive binding of ACh is ignored.