Effect of Azide on Some Spectral and Kinetic Properties of Pig-Plasma Benzylamine Oxidase

Eur. J. Biochem. 48, 237-243 (1974)

Effect of Azide on Some Spectral and Kinetic Properties of Pig-Plasma Benzylamine Oxidase Anders LINDSTROM, Bengt OLSSON, and Gosta PETTERSSON

Avdelningen for Biokemi, Kemicentrum, Lunds Universitet

(Received April 10,'July 8, 1974)

1. The effect of azide on optical absorption and electron paramagnetic resonance (EPR) spectra of pig-plasma benzylamine oxidase has been examined and correlated with the effect of azide on the catalytic activity of the enzyme.

2. A strong reversible interaction between azide and copper in benzylamine oxidase, corresponding to complex-formation with a dissociation constant less than 0.2 mM, was detected by EPR techniques. This interaction is competitive with the EPR-detectable binding of cuprizone to copper in the enzyme, but is not affected by reactions involving the active-site pyridoxal phosphate and has no evident effect on enzyme activity.

3. A weaker reversible interaction between azide and protein-bound copper, corresponding to complex formation with a dissociation constant of 40 mM, was detected spectrophotometrically and by EPR techniques. An absorption band centered at 390 nm is associated with the latter complex, the apparent stability of which was found to be unaffected by a 20-fold variation of the oxygen concentration as well as by reduction of the enzyme with substrate.

4. Azide acts as an uncompetitive inhibitor of benzylamine oxidase with an inhibition constant close to 40 mM. 100 mM azide has essentially no effect on the reactivity of the active-site pyridoxal phosphate towards phenylhydrazine or benzylamine. It is concluded that formation of the enzyme . azide complex exhibiting the 390 nm absorption band precludes reoxidation of the substrate-reduced form of the enzyme, indicating that at least one of the two copper ions in benzylamine oxidase may have a direct catalytic function in relation to the reoxidation process.

Pig-plasma benzylamine oxidase, similarly to other plasma monoamine oxidases and various diamine oxidases, contains firmly protein-bound copper and pyridoxal phosphate [l]. There is strong evidence that the function of pyridoxal phosphate in these enzymes is closely related to its ability to react with the amine substrates under Schiff-base formation [2,3], but the function of copper is not yet clear. Cupric copper has been reported to restore the activity of copper-free benzylamine oxidase [4], but addition of substrate to the enzyme under anaerobic conditions does not lead to any detectable valency change of the two cupric ions present per enzyme molecule [5,6]. The strong inhibitory action of the copper-chelating cuprizone has recently been found to be due to an interaction with enzyme-bound pyridoxal phosphate rather

Abbreviation. EPR, electron paramagnetic resonance. Enzyme. Benzylamine oxidase, monoamine oxidase, or

monoamine : 0, oxidoreductase (deaminating) (EC 1.4.3.4).

than with enzyme-bound copper [7], and azide, which is also known to have a high affinity for copper and to act as a strong inhibitor of copper-containing oxidases such as laccase and ceruloplasmin [S - 101, does not affect the catalytic activity of benzylamine oxidase and related enzymes even at a concentration of 1 mM

The latter observation could be of great interest from a mechanistical point of view, since it might indicate either that copper in benzylamine oxidase is not easily accessible to external ligands or that the binding of external ligands to copper has essentially no effect on the catalytic activity of the enzyme. It was, therefore, considered of importance to examine whether any interactions between benzylamine oxidase and azide can be detected by techniques that are not dependent upon determinations of enzyme activity.

The present investigation describes the effect of azide on opticdl absorption and electron paramagnetic

[11- 131.

Eur. J. Biochem. 48 (1974)

238 Effect of Azide on Benzylamine Oxidase

resonance (EPR) spectra of pig-plasma benzylamine oxidase. Data relating the spectrometrically detected interactions between azide and benzylamine oxidase to the effect of azide on the catalytic activity of the enzyme are also reported.

EXPERIMENTAL PROCEDURE

Materials

The preparation of homogeneous benzylamine oxidase from pig plasma and methods for determina- tion of protein concentration and assay of enzyme activity have been described previously [14]. Enzyme preparations used in the present investigation were at least 90 pure according to specific activity determinations. The phenylhydrazone derivative of the enzyme was prepared by titration with phenylhydrazine [3], excess of the carbonyl reagent being removed by dialysis. Other chemicals used were commercial samples of highest available purity. Solutions of azide were prepared immediately before use.

Met hods Unless otherwise stated, experiments reported in

the present paper were carried out at 25 "C in 0.1 M phosphate buffer, pH 7.0, saturated with air (0.25 k 0.005 mM oxygen). In some experiments the oxygen concentration was varied between 0.05 and 1.0mM by the techniques described elsewhere [3], oxygen concentrations being determined polarographically with a Clark electrode combined to an Eschweiler Combi-Analysator U.

Optical absorption spectra were recorded with a Zeiss PMQII spectrophotometer. Apparent first- order rate constants for the reaction between enzyme and phenylhydrazine, and for reduction of the enzymatic 470 nm chromophore by substrate, were determined by stopped-flow techniques as described previously [3,6]. EPR spectra were recorded with a Varian E-3 spectrometer at 77 K and about 9.13 GHz, using 90- 120 pM enzyme.

RESULTS

Effect of Azide on Electron-Purumugnetic-Resonance Spectra oj Benzylamine Oxidase

Fig. 1 A shows a low-temperature EPR spectrum at 9.13 GHz of pig-plasma benzylamine oxidase dissolved in 0.1 M phosphate buffer at pH 7. The spectrum agrees in all essential features with those reported previously [5,6], and can be reasonably well simulated by assigning an identical set of EPR

I I I I I

2600 2800 3000 3200 3400 Magnetic f ie ld (gauss)

Fig. 1. EiIect of azide on the EPR spectrum of' henzylamine oxiduse. Spectra were recorded at 77 K and 9.13 GHz using 90 pM enzyme dissolved in 0.1 M phosphate buffer, pH 7, containing 0 (A), 0.10 (B), 0.22 (C), 0.53 (D), and 1.2 (E) mM azide

parameter values (g, , = 2.08; A = 155 gauss; g , = 2.06; A , = 15gauss) to the two cupric ions in the protein. At high resolution, several superhyperfine lines with a splitting of approximately 14 gauss can be detected on the main peak, presumably arising from nitrogenous ligands in the protein [15].

In the presence of increasing concentrations of azide, the EPR spectrum of benzylamine oxidase gradually changes towards that obtained with about 1 mM azide (Fig. lE), which exhibits a completely different hyperfine structure in the ,gl, -region (g , , = 2.25; A = 165 gauss) and an obvious superhyperfine pattern of about nine regularly spaced lines on the main peak with a splitting of 14 gauss. No further EPR spectral changes were observed on raising the azide concentration to 6 mM (Fig. 2A), and dialysis of samples incubated with 6 mM azide restored the original spectrum of the protein (Fig. 2B).

Although it cannot be excluded that the two copper ions in benzylamine oxidase give rise to slightly different overlapping EPR signals, the possibility that 1 mM azide affects only one of these signals seems most unlikely in view of the apparent homo- geneity of the spectrum in Fig. 1 E and the large shift of the hyperfine lines at low field in this spectrum.


A. Lindstrom, B. Olsson, and G. Pettersson 239

I

I I I I I I

I I I /

2600 2800 3000 3200 3400 Magnet ic f ie ld (gauss)

Fig. 2. Eflect of azide on the EPR spectrum of henzylamine oxiduse. Spectra were recorded at 77 K and 9.13 GHz using 90 pM enzyme dissolved in 0.1 M phosphate buffer, pH 7, containing 6 (A) and 140 (C) mM azide. Spectrum B was obtained after dialysis of the protein sample treated with 6 mM azide

Further evidence that both copper signals are essentially identically affected by azide is given by the hetero- geneity of the spectra recorded at azide concentrations below 1 mM (Fig. 1 B-D), which exhibit mixed contributions from the two extreme spectra A and E rather than a continuous conversion of spectrum A into spectrum E. Approximately equal contributions of the two extreme spectra are obtained at a total azide concentration of about 0.25 mM. Assuming that the EPR spectral changes observed can be attributed to a non-cooperative and equally firm binding of one azide ion per each copper ion, the latter datum would correspond to a dissociation constant of about 0.15 mM for the complex formed between azide and enzyme-bound copper; it may be observed that concentrations of free ligand are considerably lower than the total concentrations of azide indicated in Fig. 1. A slightly larger estimate of the dissociation constant (0.20 mM) is obtained under the assumption that the EPR spectral changes are due to the binding of a single azide ion, a possibility that cannot be definitely excluded.

At very high concentrations of azide, an additional interaction between enzyme and ligand can be detected by EPR techniques. Above 10mM azide the EPR spectrum of the protein thus gradually changes towards that obtained in the presence of 140mM azide (Fig. 2C), which is characterized by a further

I I I I

I I

/- I

I I I

v 2600 2800 3000 3200 3400

Magnet ic f ield (gauss)

Fig. 3. Eflect of'azide on the EPR spectrum of substrate-reduced henzylamine oxidase, and of the phenylhydrazone derivative of the enzyme. Spectra were recorded at 77 K and 9.13 GHz using 120 pM protein dissolved in 0.1 M phosphate buffer, pH 7, containing 0 (A), 0.21 (B), and 0.80 (C and D) mM azide. Spectrum D was obtained with native benzylamine oxidase after anaerobic reduction of the enzyme with 5 mM benzylamine, and spectra A - C with the phenylhydrazone derivative of the enzyme

upfield shift of the first two hyperfine lines at low field. The latter interaction was also found to affect the absorption spectrum of the protein, and could be more conveniently examined by spectrophotometric techniques.

Effect of A z ide on Electron-Paramagnetic-Resonance Spectra of Benzylamine Oxidase Derivatives

Treatment of benzylamine oxidase with hydrazines leads to the irreversible formation of inactive enzyme species which most likely represent hydrazone derivatives obtained through reaction with the protein- bound pyridoxal phosphate [3]. Fig. 3A shows that the EPR spectrum of the phenylhydrazone derivative of benzylamine oxidase is essentially identical with that of native enzyme, and Fig. 3B and 3C illustrate that low concentrations of azide affect spectra of the phenylhydrazone derivative in the same way as those of native enzyme. It was, similarly, found that reduction of the enzyme with a large excess of substrate ( 5 mM benzylamine) under anaerobic conditions had no significant effect on the EPR-detectable interaction between enzyme-bound copper and low concentrations of azide (Fig. 3 D).

Eur. J . Biochem. 48 (1974)


I I I I

\i 2600 2800 3000 3200 3400

Magnetic field (gauss)

Fig. 4. Competition between azide and cuprizone fo r the phenylhydrazone derivative of benzylamine oxidase. EPR spectra were recorded at 77 K and 9.13 GHz using 120 pM protein dissolved in 0.1 M phosphate buffer, pH 7, containing 4mM cuprizone and 0 (A), 0.2 (B), 1.3 (C), 3.8 (D), and 8.6 (E) mM azide

Competition between Azide and Cupvizone for Copper in Benzylamine Oxidase

A recent investigation has provided evidence that the inhibition of benzylamine oxidase by the copper- chelating reagent cuprizone can be attributed to an interaction with enzyme-bound pyridoxal phosphate, but the results obtained also indicated that cuprizone binds to copper in the protein [7]. The addition of cuprizone to the phenylhydrazone derivative of benzylamine oxidase was thus found to result in pronounced EPR spectral changes, the most characteristic of which is the appearance of a new line centered around 3060 gauss (Fig. 4A).

Fig. 4B-E show that the intensity of this line is gradually suppressed in the presence of increasing concentrations of azide, the spectrum obtained with a large excess of azide being indistinguishable from that obtained under similar conditions in the absence of cuprizone. The azide concentration required to produce a spectrum in which approximately half of the signal derives from the enzyme-azide component appears to be in the order of 3 mM, indicating that the presence of 4 mM cuprizone decreases the apparent affinity of enzyme-bound copper for azide by a factor

0.6

0.4

8

e m Lo D <

0 .2

0 .

I I 1 400 500 600

Wavelength (nrn)

Fig. 5. Effect ojazide on the absorption spectrum ofbenzylamine oxidase. 58 pM enzyme in 0.1 M phosphate buffer, pH 7, containing varied amounts of azide. The dashed curve indica- tes the difference spectrum obtained in the presence of 140 mM azide

of about 20. Since the EPR-detectable interaction between enzyme and cuprizone has been reported to correspond to a dissociation constant of approximately 0.2 mM [7], the above observations seem to be both qualitatively and quantitatively consistent with a competitive binding of cuprizone and azide to copper in benzylamine oxidase.

Effect of Azide on Optical Absorption Spectra of Benzyiamine Oxiduse

While 3 mM azide has no significant effect on the ultraviolet and visible absorption spectrum of benzylamine oxidase dissolved in 0.1 M phosphate buffer at pH 7, the addition of higher concentrations of azide causes a new absorption band centered at 390 nm to appear (Fig. 5). The 390 nm absorbance changes occurring on mixing 10 pM enzyme with 50 mM azide were found to be too rapid to be examined by stopped-flow techniques. Dialysis of enzyme solutions treated with 140mM azide restored the original absorption spectrum of the protein.

The variation with azide concentration of the 390 nm absorbance ( A ) is shown in Fig. 6, and is consistent with the reversible formation of an enzyme . azide complex according to the relation

(1) K E + N;* E-N;


A. Lindstrom, B. Olsson, and G. Pettersson 24 1

I

-0 50 100 150 [Azide] (mM)

Fig. 6. Spectrophotornetric titration of benzylamine oxidase with azide. Plot according to Eqn (2) of the data in Fig. 5. A stands for the 390 nm absorbance as the actual azide concentration, A, and A , for the values obtained at zero and infinite concentrations of azide, respectively

which predicts that

Table 1. EfJect of azide on the reactivity ofpyridoxalphosphate in benzylamine oxidase Apparent first-order rate constants for formation of the phenylhydrazone derivative of benzylamine oxidase (kp), and for reduction of the enzyme by substrate (ks), were determined at 25 "C from the time course of the 430 nm (470 nm) absorbance changes occurring on mixing about 5 pM enzyme with 0.5 mM phenylhydrazine (5.0 mM benzylamine) in 0.1 M phosphate buffer, pH 7, containing varied amounts of azide

Concn of azide kP ks

mM S - '

0 1

30 100

5.6 & 0.4 6.2 rfr 0.6 5.3 rfr 0.6 4.4 & 0.6

6.0 0.5 6.2 rfr 0.5 5.4 rfr 0.6 5.8 & 0.6

30

1 . c

10

where A, and A , stand for the 390 nm absorbances at zero and infinite concentrations of azide, respectively. The estimate of the dissociation constant K obtained by regression analysis of the data in Fig. 6 was K = 40 (k 5 ) mM, and the difference A , - A, was found to correspond to an absorption coefficient of 5.2 (k 0.5) mM-' x cm-' for the 390 nm chromophore. The latter value agrees well with the absorption coefficient of 4.6 mM-l x cm-' reported for the 390 nm chromophore appearing on the binding of azide to type 2 copper in ceruloplasmin [lo].

The above experiments were performed in buffer solutions saturated with air (0.25 mM oxygen). Varia- tion of the oxygen concentration from 0.05 to 1 .O mM did not significantly affect the estimates of K obtained on spectrophotometric titration of the enzyme with azide at 390 nm, indicating that there is no competition between oxygen and azide for the oxidized form of the enzyme. It was, similarly, found that reduction of the enzyme by substrate under anaerobic conditions, or formation of the phenylhydrazone derivative of the protein, had no significant effect on the stability of the enzyme . azide complex exhibiting the 390 nm absorption band.

I [Azide] (mM)

I I I I I 0 2 4 6 8 10

11 [ Benzylamine] (mM-') Fig. 7. Effect of azide on the catalytic activity of benzylamine oxidase. Lineweaver-Burk plots for the enzymatic oxidation of benzylamine at 25 "C in 0.1 M phosphate buffer, pH 7, containing varied amounts of azide, v = molar enzymatic reaction rate

Effect of Azide on the Catalytic Activity of Benzylamine Oxidase

1 mM azide has no significant effect on the steady- state rate of oxidation of benzylamine by pig-plasma benzylamine oxidase in 0.1 M phosphate buffer at pH 7, neither does it affect the rate of hydrazone formation between phenylhydrazine and enzyme- bound pyridoxal phosphate or the rate of reduction of the enzymatic 470 nm chromophore by substrate (Table 1). As shown by the Lineweaver-Burk plots in Fig. 7, however, the presence of higher concentra-



0 0 20 40 60 80

[Az ide ] (rnM)

Fig. 8. Replots of intercepfs of the linear graphs in Fig. 7 vs the concentration of azide

tions of azide results in an uncompetitive inhibition of the enzymatic activity during steady-state. Experi- mental points in a replot of intercepts of the linear Lineweaver-Burk graphs vs the azide concentration (Fig. 8) fall well along a straight line corresponding to an inhibition constant of 40 mM, i.e. agreeing with the dissociation constant for the enzyme . azide complex exhibiting the 390 nm absorption band. Examina- tion of the rate of formation of the phenylhydrazone derivative of benzylamine oxidase (and of the rate of reduction of the enzymatic 470 nm chromophore by substrate) in the presence of high concentrations of azide established that the inhibitory effect of azide cannot be attributed to a decreased reactivity of the enzyme-bound pyridoxal phosphate (Table 1).

DISCUSSION

Treatment of pig-plasma benzylamine oxidase with high concentrations of the copper-chelating reagent diethyldithiocarbamate has been reported to result in the formation of an inactive protein depleted of copper [4], but no information has previously been available as concerns the effect on enzyme activity of ligand-binding to copper while still being protein-bound. Recent attempts to obtain such information from inhibition studies involving cuprizone were unsuccessful, as the latter copper-chelating reagent was found to react irreversibly with the active- site pyridoxal phosphate [7]. On the other hand, examination of the effect of cuprizone on EPR spectra of the enzyme and its phenylhydrazone derivative led to the conclusion that external ligands do interact with copper in benzylamine oxidase. The present investigation lends strong support to this conclusion in showing that the presence of low concentrations

(less than 1 mM) of azide leads to EPR spectral changes which, at present, can be best understood in terms of a reversible binding of azide to both copper ions present in the protein. This interaction is strictly competitive with the EPR-detectable binding of cuprizone, but does not appear to be affected by reactions involving the active-site pyridoxal phosphate and has no significant effect on the catalytic activity of the enzyme.

The present results, therefore, seem to establish that external ligands such as azide and cuprizone may be firmly bound to copper in benzylamine oxidase without any evident concomitant effect on enzyme activity. This observation could, obviously, be taken to indicate that copper has no direct catalytic function in the enzyme, but examination of the effect of high concentrations of azide on the spectral and kinetic properties of the enzyme provides evidence in the opposite direction. The reversible formation of an inactive enzyme . azide complex with a dissociation constant of about 40 mM can thus be inferred from the data presented in Fig. 5 - 8, and the EPR-spectral and 390 nm absorbance changes associated with the formation of this complex strongly suggest that azide is bound as a ligand of copper. Although no firm conclusion can be drawn as to the detailed stoichio- metry of the spectrometrically detected interactions between enzyme and azide, it may be noted that similar 390 nm absorbance changes have been reported to occur on the binding of azide to a single copper ion in ceruloplasmin (type 2 copper, the binding charac- teristics of which appear to be analogous to those of copper in benzylamine oxidase) [lo]. The observation that absorption coefficients for the 390 nm chromo- phores appearing on azide-binding to ceruloplasmin and benzylamine oxidase are of closely agreeing magnitude might, therefore, suggest that only one of the two copper ions in benzylamine oxidase is involved in the latter interaction. This would be consistent with the observed linearity of the Dixon plot in Fig. 8, which provides evidence that a single azide ion per active site is sufficient to inhibit the enzyme. Benzyl- amine oxidase has been shown to contain a single active site per protein molecule [14,16], and the possibility that only one of the copper ions is catalytically important cannot be excluded.

Steady-state kinetic data for benzylamine oxidase have previously been shown to be consistent with the minimal kinetic scheme

where enzyme (E) and substrate (S) react rapidly to give a reduced enzyme species (E') which is sub-


A . Lindstrom. B. Olsson. and G. Pettersson 24 3

sequently reoxidized in a rate-limiting process involving the participation of molecular oxygen [6]. When interpreted in view of Scheme (3), the observation that azide acts as an uncompetitive inhibitor of the enzymatic reaction during steady-state is consistent with the formation of an inactive complex between azide and the reduced form of the enzyme according to the relation

E‘ + N; $ E‘-N, (4)

and the kinetic data in Fig. 8 indicate that the dissociation constant for complex formation according to reaction (4) agrees with that of the enzyme . azide complex exhibiting the 390 nm chromophore. The latter result provides evidence that the inhibition of enzyme activity and the 390 nm absorbance changes observed with the “free” enzyme (Reaction 1) are due to the same interaction between azide and protein- bound copper, reduction of the enzyme by substrate having no significant effect on the affinity of protein- bound copper for azide. Assuming that such is the case and that the binding of azide is rapid in compari- son to the rate-limiting reoxidation process, conditions which appear to prevail according to the present results, it can be shown that the combination of azide to the “free” enzyme is kinetically insignificant, i.e. that it leads to an uncompetitive inhibition pattern indistinguishable from that obtained under the assumption that azide combines exclusively to the reduced form of the enzyme.

Interpretation of the inhibition data in view of the simplified reaction mechanism expressed by Scheme (3), therefore, leads to the conclusion that formation of the enzyme . azide complex exhibiting the 390 nm absorption band has no inhibitory effect on the reduction of the enzyme by substrate, but prevents reoxidation of the substrate-reduced form of the enzyme. The results listed in Table 1 lend support to this idea in showing that the reactivity of the active-site pyridox- a1 phosphate remains essentially unaffected in the presence of 100 mM azide. On the other hand, the observation that variation of the oxygen concentration by a factor of 20 has no significant effect on the apparent stability of the 390 nm chromophoric enzyme . azide complex makes it uncertain whether the inhibitory action of azide can be attributed simply to a competition between azide and oxygen for copper in the enzyme.

Summing up the above discussion, the present results show that the EPR-detectable strong interaction between azide and copper in pig-plasma benzylamine oxidase has no evident effect on the catalytic properties of the enzyme, whereas the weaker binding of azide to copper leading to the 390 nm absorbance changes precludes reoxidation of the substrate-reduced form of the enzyme. The latter observation provides strong evidence that at least one of the two copper ions in benzylamine oxidase has a catalytically important function, presumably participating in the sequence of reactions by which the reduction of molecular oxygen is facilitated. Experiments with the objective of elucidating the precise role of copper in this process are in progress.

This investigation was supported by grants from the Swedish Natural Science Research Council.

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A. Lindstrom, B. Olsson, and G. Pettersson, Avdelningen for Biokemi, Kemicentrum, Lunds Universitet, Box 740, S-220 07 Lund 7, Sweden

Eur. J . Biochem. 48 (1974)

Effect of Azide on Some Spectral and Kinetic Properties of Pig-Plasma Benzylamine Oxidase

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Transcript of Effect of Azide on Some Spectral and Kinetic Properties of Pig-Plasma Benzylamine Oxidase