2. Methods
2.6 Activity Assay
Amine oxidase activity was measured spectrophotometrically by monitoring H2O2 production through a coupling assay by horseradish peroxidase (HRP) using DMAB (3-dimethyl-aminobenzoic acid) and MBTH (3-methyl-2-benzothiazolinone hydrazone) as substrates (Stoner, 1985). A DMAB-MBTH conjugated purple indamine dye formed
during reaction can be measured at absorbance of 595 nm. Assays were carried out in a reaction buffer containing 50 mM sodium phosphate buffer, pH 7.4, 2 µg enzyme and 100 µM amine substrate in a total volume of 0.5 mL at 30 ℃ for 30 sec or longer. The detection buffer contains 2.5 U horseradish peroxidase, 2 mM DMAB, 0.04 mM MBTH in a final volume of 0.5 mL. Blank assay medium did not contain
substrates. The Cu2+-deficient inactive enzyme was incubated at 30 ℃ for 30 min with 50 mM CuSO4 in 50 mM sodium phosphate buffer (pH 6.8), before subjecting activity assay. The enzymatic reaction was started by mixing substrate and AGHO in a reaction buffer at room temperature for 30 sec. The reaction was stopped by adding 0.5 mL detection buffer, mix well and measure the colored product at
absorption of 595 nm. Absorbance measurement were obtained with a
Hitachi U-3010 at 30 ℃ using quartz cuvettes with 1 cm path length.
2.7 H2O2 Standard Curve
The procedure is the same as that of activity assay except that amine oxidase and substrates were replaced with H2O2 in amounts from 1.0 to 20 nmoles. The standard curve of H2O2 concentration and O.D.
was illustrated in Fig. 1.
2.8 Kinetic Measurement
Substrate stock solutions were freshly prepared in distilled water or methanol. The concentration of substrate in the kinetic study varies based on type of substrate due to substrate inhibition effect. The reaction time was 30 sec for most of amine substrates except
benzylamine, phenylpropylamine, tryptamine, and D, L-octopamine.
The oxidation of substrates was determination by coupled assay (as described in Activity Assay). The data were fitted non-linearly by at least six or more substrate concentrations. The curve fitting was performed using Michaelis-Menten equation on the SIGMA plot
program Enzyme Kinetics Module 1.1. Those substrates demonstrating substrate inhibition were also using Eq. 3:
V=Vmax / (1+Km/[S] + [S]/Ki) (3)
2.9 Electrophoresis and Redox-Cycling Staining
All SDS PAGE was performed with a Bio-Rad Mini Protein II apparatus. 10% polyacryamide gels were prepared. Enzyme samples were boiled for 5 min in the presence of 5% 2-mercaptoethanol before loading onto the SDS-polyacryamide gel. After electrophoresis, the gel was stained with Coomassie Blue. Standard proteins for calibration (kDa) were MBI marker #SM0431.
For redox-cycling staining, proteins separated by 10%
SDS-polyacryamide gels were electroblotted on to a 0.45 µm
nitrocellulose membrane in an ice-cold transfer buffer (25 mM Tris, pH 8.3, 192 mM Glycine, 20% (v/v) methanol) at a constant current of 200 mA for 2 h. The nitrocellulose membrane was then immersed in the Glycinate/NBT solution (0.24 mM Nitro Blue Tetratzolium in 0.1 M potassium glycine , pH 10) at 25 ℃for 30-45 min in the dark. The quinoproteins would be stained as blue-purple bands on the membrane.
2.10 Phenylhydrazine Titrations
Reactive TPQ in AGHO or its mutants can be quantified by the titration with phenylhydrazine, which forms a stable, intensely yellow-coloured adduct with a maximum absorption at λ≈ 438 nm (Choi et al., 1995). The phenylhydrazine stock solution (1 mM) was prepared fresh by dissolving phenylhydrazine hydrochloride in distilled water and storing it at 4 ℃ in the dark before using. TPQ titration of
AGHO was carried out by step-wise adding phenylhydrazine stock solution in 1 ml of enzyme (12.5 µM) to the final concentrations of 0, 1.0, 2.0, 4.0, 6.0, 10.0, 12.0, 13.0, 15.0, and 17.0 µM. Each spectrum was recorded 15 min after incubation at 30 ℃ following each addition.
Results
Expression and Purification of Wild-type and Mutant Histamine Oxidases— The production of an active quinone-containing form of CAOs is dependent on the presence of Cu2+ ions. We prepare both the Cu2+-deficient inactive precursor of AGHO and the Cu2+-containing active enzyme. The purified AGHO (Cu2+-containing active form) exhibited a brownish pink color, whereas the Cu2+-free form inactive enzyme was colorless. Although the enzyme activity under two different expression conditions (see Methods 2.4) seems different, the
purification efficiency is the same (Table.3). The Y316A, Y316E, Y316F, and Y316H mutants were also purified as a copper/TPQ-free, inactive form to homogeneity as indicated by SDS-polyacryamide gel electrophoresis (Fig. 2).
Titration with Phenylhydrazine— The presence of a quinone moiety in the A. globiformis histamine oxidase was investigated by reaction with phenylhydrazine (Paz et al., 1991). The TPQ biogenesis in the
Cu2+-reconstituted enzyme was titrated with the carbonyl reagent phenylhydrazine, which reacts with the topaquinone to form a yellow adduct. The enzyme solution was incubated with phenylhydrazine at 30
℃ for 15 min, the full spectrum of the enzyme was scanned and recorded. The TPQ in AGHO was titrated with 0~17 µmole
phenylhydrazine. An absorption peak at λmax= 438 nm increased with increasing the concentration of phenylhydrazine (Fig. 3A). The
endpoint of titration was approximated at about ~ 12 nmole (Fig. 3B).
Upon calculation, the ratio of TPQ: AGHO monomer is about 0.5:1.0.
Relative Activity of AGHO with Various Substrates—Although histamine was reported to be the primary substrate for AGHO, other amines were also indicated to be catalyzed by this enzyme (Shimizu, 1994). To confirm this observation, the reactivity of Cu2+-reconstituted active AGHO to various, natural or Xenobiotic amines (Appendix. 1) were treated. The relative activities (compared with histamine) of other amines at 0.1 mM are listed in table 3. Accordingly, AGHO exhibited higher reactivity to phenylethylamine (154%), tyramine (107%) dopamine (125%) than to the histamine. AGHO exhibited moderate activity to some amines, including 2,3-dihydroxypenylethylamine (84%), 2,4-dihydroxypenylethylamine
(80%), 3-methoxyphtnylethylamine (67%) and 4-methoxy- phenylethylamine (55%). AGHO showed a little or no activity to
benzylamine (0.5%), serotonin (12%), Norepinephrine (4%), and all the aliphatic amines studied (Table. 3). Notably, all amines except
histamine exhibit strong to moderate inhibition at higher concentration.
Kinetic studies of AGHO with Histamine and its derivative—Although CAOs exhibit common mechanistic features, the substrate specificities of these enzymes appear to be different. The amine oxidases from
bacteria show a preference for aromatic amines. In this work, we tend to investigate the structural characteristics of various amines that may influence their binding affinity to AGHO. Tables 4-6 show the kinetic constants (Km, Kcat, Kcat/Km) of AGHO to various structurally related amines. Initial rates of AGHO at each concentration of corresponding amine substrate were determined at 30 ℃, pH 7.0 for 0-10 min. The
non-linear curve of initial rate vs. substrate concentration was fitted to the appropriate Michaelis-Menten equation or to Eq. 3. The plots of initial rate vs. substrate concentration for various amines were shown in Fig. 4-9. The results show that almost all amines (except histamine) exhibit strong to moderate substrate inhibition to recombinant AGHO.
The substrate inhibition effect was not significant in histamine oxidation.
As shown in Table 5, AGHO exhibited slightly increasing Km values with increasing hydroxyl groups on the aromatic ring of the
phenylethylamine derivatives. Recombinant AGHO shows a preference to more hydrophibic amines, as indicated by the Kcat/Km, a specificity constant, for phenylethylamine (1.11), tyramine (0.77), 2,3-Dihydroxy- phenylethylamine (0.54), 2,4-Dihydroxyphenylethylamine (0.53), and 3,4-dihydroxyphenylethylamine (0.42)(Table 5). The result suggests that Kcat/Km decrease with decreasing hydropathy.The Km for histamine was about twenty seven times higher than that of tryptamine.
Next, we want to study the effect of different chain length connecting amine group and aromatic ring to catalytic activity of AGHO. Benzylamine, phenylethylamine, phenylpropylamine, and phenylbutylamine were chosen in this study (Table 6). As shown Table 6, KM values of amines decrease in nearly linear fashion with increasing chain length of the alkyl carbon chain connecting amine and aromatic groups. The catalysis of AGHO for benzylamine and
phenylpropylamine was slower. However, the Kcat decrease drastically with increasing the alkyl chain length. This postulation was confirmed by the kinetic studies of 2 more hydrophobic methoxyl derivatives of phenylethylamine, 3-methoxy-phenylethylamine (Km=15.97 ± 1.76,
Kcat=16.67 ± 0.99, Kcat/Km=1.04) and 4-methoxy-phenylethylamine (Km=16.73 ± 3.63, Kcat=17.60 ± 2.20, Kcat/Km=1.05). The decreasing substrate preference was with increasing hydroxyl group numbers on the benzene ring of the aromatic amines, indicating the binding affinity is suppressed with the increasing the hydrophilicity of aromatic amines.
However, the effects of methoxyl group substituted in the para and inter position increase their inhibitory potency as indicated by e Ki values for 3-methoxy-phenylethylamine and 4-methoxy-phenylethylamine, were 56.69 ± 5.74 µM, and 32.18 ± 5.90 µM, respectively. The oxidation rate of aliphatic amine were too slow to determinate the kinetic parameters.
Catalytic properties of Cu2+-reconstituted enzymes—Y316, at in the end of the substrate channel, is considered as a “gate” for substrates to access TPQ. It is hypothesized that it may play a key role in mediating the substrate specificity of many CAOs. All five Y316 mutants can be purified as wild type AGHO dose; however, mutations at Y316 alter amine oxidase activity (Table. 2). After incubation with 50 µM CuSO4
at 30 ℃ for 30 min, three Y316 AGHO mutants, Y316A, Y316E, and Y316H, showed no catalytic activity toward aromatic amines, including histamine, phenylethylamine, or tyramine, as well as aliphatic amines, including methylamine or ethylamine. The specific activity of AGHO Y316F decreased 75%, compared with that wild-type enzyme in the presence of 0.1 mM histamine. Interestingly, the TPQ biogenesis of all AGHO Y316 mutants were not altered as illustrated in Fig. 10 and Fig.
11.
In vitro reconstitution with 1 mM copper did not influence the specific activity of Cu2+-reconsituted wild-type enzyme. Even the
enzyme was incubated with 50 µM CuSO4 at 4 ℃ for 24 hr, the activity of enzyme did not increase. The wild-type and Y316 mutants could be completely reconstituted to active enzyme when incubation with 50 µM CuSO4 at 30 ℃ for 30 min.
Discussion
In this study, we have demonstrated that the oxidative modification of the precursor tyrosine to TPQ could be completed in the presence of 50 µM CuSO4. According to the result of redox-cycling staining, wild type AGHO was active as a result of the formation of TPQ by a
self-processing mechanism. The storage time of the apo-form AGHO at 4 ℃was longer then Cu2+-containing active form AGHO. This is
probably because Cu2+ can induce a oxidative damage on the enzyme due to its strong oxidative catalytic activity.
The correlations between specific activity and titratable TPQ have been described by Klinman and co-workers (Janes et al., 1991; Cai et al., 1994). It is presently unclear whether low TPQ to CAO ratio (normally around 0.6) is due to low TPQ-to-copper ratios reflect incomplete organic cofactor biogenesis or due to the conformations of the matured enzymes in which the quinone cofactor is inaccessible to phenylhydrazine.
It has long been recognized that CAOs generally oxidize a variety of primary amines. For example, AGAO oxidizes a range of aromatic, alkyl, and aliphatic primary amines. In the case of PSAO, the preferred substrates are primary diamines, such as putrescine and cadaverine. The HPAO preferentially catalyzes oxidation of the aliphatic amines, such as methylamine and ethylamine.
In this work, we showed that AGHO prefer hydrophobic amines as its substrates. The Kcat/Km values of hydrophilic amines, including dopamine or 3,4-dihydroxyphenylethylamine (Kcat/Km=0.42), tyramine
or 3-hydroxyphenylethylamine (Kcat/Km=0.77), 2,3-dihydroxy-
phenylethylamine (Kcat/Km=0.54), and 2,4-dihydroxyphenylethylamine (Kcat/Km=0.53), were lower than that of hydrophobic amines, such as phenylethylamine (Kcat/Km=1.11), 3-methoxyphenylethylamine (Kcat/Km=1.04), and 4-methoxypheylethylamine (Kcat/Km=1.05).
Substitution has a modest effect on Kcat. The hydrophobicity is probably the determinant in substrate to affect its binding to AGHO, implying the presence of a lipophilic binding pocket at the active site of AGHO (Wilce et al., 1997). The relationship between the hydropathy and the Kcat/Km value of amine were demonstrated in this study. Although the hydropathy of the amines studied in this work is unknown, the scold of corresponding amino acids of these amines, such as histamine,
phenylethylamine, tyramine, and tryptamine, may provide a reference about the hydropathy of the amines (Plaecz, 2002). From the analysis of lipophilicity scales, phenylalanine (with a scold of 179) is more
hydrophobic than that of Trp (147), Tyr (64), and His (-34). The Kcat/Km
value of those amines follows the same fashion to the hydropathy of the corresponding amino acids. This result indicates that hydrophobicity of the amines is one of the essential factors that mediate the binding and recognition of amines by the AGHO.
The substrate specificity of CAOs is the challenging topic. Since amine substrates are recognized in their protonated, positively charged form, the electrostatic potential and surface topology at the entrance to the channel may be important for substrate recognition. In the previous inhibition study (Shepard et al., 2002), 1,4-diamino-2-chloro-
2-butene and 1,6-diamino-2,4-hexadiyne effectively inhibit six amine
oxidases (AGAO, ECAO, PPLO, PSAO, BPAO, and EPAO).
Distinctions among the active sites must be responsible for
differentiating the chemical interactions between the inhibitors and enzymes. With the crystal structures of amine oxidases from different source, structure-like substrate or inhibitor study might be combined with bioinformatics tool for the study of substrate specificity.
Based on the clinical investigation, the determination of SSAO activity in mammalian might be a candidate biochemical marker for screening healthy people with high risk of atherosclerosis for the
presence of early atherosclerotic lesions. The amine content of fish can food is the determination of fresh order. The clarification of substrate specificity among different amine oxidases is helpful for biosensor application and medical drug design.
AGHO shows a clear substrate preference for aromatic amines. It is also apparent that substrate specificity of AGHO depends on the hydrophobicity of amines and the length of alkyl amine on the aromatic ring.
Phenylethylamine is a good substrate for AGHO. Two derivatives, benzylamine and phenylpropylamine, also contain a benzene ring but with different length of alkyl amine group are poor substrates for AGHO. AGHO shows a slow catalytic activity to benzylamine in our assay conditions. The aliphatic amine, such as methylamine or
ethylamine, also have slow reaction rate. Structural comparison of benzylamine, phenylethylamine, methylamine, and ethylamine revealed that the main differences were phenolic ring. It is reasonable to assume that there was existence of a cavity of active site holding aromatic ring
of amines for catalytic reaction. The appropriate chain length of alkyl carbon chain determines good substrate or not.
The amino acid sequence alignment (Appendix. 7) shows that the residues acted as “gate” were Phe298 in PSAO, Tyr381 in ECAO, Tyr296 in AGAO, Ala317 in HPAO, and Y316 in AGHO. In the
structure of native ECAO, the active site is buried with no obvious entry route for the monoamine substrates. In the crystal structure of ECAO complex with 2-HP (Wilmot et al., 1997), the pyridine ring of 2-HP is almost completely buried and has displaced Tyr381, which forms a π/π ring-stacking interaction with the pyridine ring. All these differences could contribute to the molecular basis for substrate specificity among these enzymes. We show that the role of Y316 could influence the reaction of enzyme and substrates. The Y316 mutant enzymes purified to homogeneity in the apo forms, and could be also activated at excess Cu ions. The activity assay of Y316 mutant enzymes show that the Y316 could act like a “gate” but was not responsible for substrate
specificity. The replacement of a bulky functional residue, tyrosine, by a nonfunctional or positive charge group, such as alanine, histamine, and glutamine, need more investigation to clarify how it cause the loss of activity.
In conclusion, we have shown here that the mutation of Y316 residue influence the catalytic activity. In this studies, the substrate specificity of Cu2+-reconstituted active recombinant AGHO have revealed that hydrophobicity is one of the essential factors that determined the affinity of AGHO to its substrates.
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