Kinetics of the Interaction between Pig-Plasma Benzylamine Oxidase and Substrate

Eur. J. Biochem. 35,70-77 (1973)

Kinetics of the Interaction between Pig-Plasma Benzylamine Oxidase and Substrate

Anders LINDSTROM, Bengt OLSSON, and Gosta PETTERSSON Avdelningen for Biokemi, Kemicentrum, Lunds Universitet

(Received December 6, 1972/February 2, 1973)

1. The rapid absorbance changes in the visible region observed on mixing pig plasma benzylamine oxidase with substrate under anaerobic conditions are due to reduction of a single chromophore, centered around 470 nm, in a single reaction phase reflecting a rate-limiting combination of substrate to the enzyme. The second-order rate constant of I s-l mM-1 for this process is unaffected by the presence of oxygen.

2. Under aerobic conditions, addition of substrate to the enzyme leads to 470-nm absorbance changes exhibiting a biphasic time-dependence. A rapid phase of transient formation of the enzyme - substrate complex is followed by a slow readjustment of the steady-state level of free enzyme, caused by substrate and oxygen consumption during the reaction.

3. The kinetic data obtained provide strong evidence that substrate-binding to benzylamine oxidase is practically irreversible. They further indicate the presence of a single kinetically significant substrate- and oxygen-independent rate-limiting reaction step in the catalytic mechanism, with a velocity constant in the order of 1 s-l.

4. A valence change of enzyme-bound copper could not be detected a t any stage of the reaction between benzylamine oxidase and substrate, irrespective of whether or not oxygen was present in the reaction solutions.

With respect to prosthetic groups, there are two types of amine oxidases in animals. The mitochon- drial type does not contain pyridoxal phosphate, whereas both plasma amine oxidases and diamine oxidases do [l]. The latter two groups of amine oxidases are unique among pyridoxal-phosphate- containing enzymes, because they require copper for activity and use 0, as a reactant. It seems most likely that a major role of copper in these enzymes is to facilitate the reduction of 0,, and that the function of pyridoxal phosphate, as with other pyridoxal phosphate enzymes, is closely related to its ability to react with the amino group of substrates under formation of a SchifF base.

No clear picture of the actual reaction mechanism has, however, emerged from the extensive studies of copper-pyridoxal-containing amine oxidases carried out during the last decade. Even though results of steady-state kinetic investigations consistently indicate a ping-pong type of mechanism [2-51, there is conflicting evidence as to the nature of the product

Enzymes. Benzylamine oxidase, monoamine oxidase or monoamine : 0, oxidoreductase (deaminating) (EC 1.4.3.4) ; diamine oxidase or diamine : 0, oxidoreductase (deaminating) (EC 1.4.3.6).

first formed [5 ] . Similarly, there is conflicting evidence as to whether or not copper undergoes a reduction-oxidation cycle during the catalytic process

Indications that a valence change of copper may take place during catalysis were obtained from stopped-flow kinetic experiments with pig kidney diamine oxidase [9], the only copper-pyridoxal- dependent amine oxidase to which rapid-reaction techniques has hitherto been applied. Similar experiments have now been performed with pig plasma benzylamine oxidase, partly in order to obtain comparative mechanistic information referring to a monoamine oxidase, and partly in order to provide a more extensive analysis of the kinetics of substrate binding to a copper and pyridoxal-phosphate- dependent amine oxidase.

[6-91.

MATERIALS AND METHODS

Materials The preparation of homogeneous benzylamine

oxidase and methods for determination of protein concentrations and assay of enzyme activity have

Vol.35, No.1, 1973 A. LINDSTROM, B. OLSSON, and G. PETTERSSON 71

been described previously [lo]. All kinetic experiments reported in the present investigation were performed a t 25 “C in 0.1 M phosphate buffer pH 7.0 using enzyme preparations of a t least goo/, purity according to specific activity determinations. Other reagents used were of analytical grade.

Steady-State-Kinetic Methods

Steady-state velocity parameters for the enzymatic reaction between benzylamine and oxygen were determined by statistical analysis of a t least tripli- cate observations of the reaction rate a t various combinations of substrate concentrations, obtained from two different sets of experiments. In the first series of experiments the benzylamine concentration was varied between 0.1 and 10 mM a t a fixed oxygen concentration of 0.25 (i 0.003) mM or 1.25 (+O.OOS) mM (air- and oxygen-saturated solutions, respectively), reaction velocities being determined spectro- photometrically from the initial rate of benzaldehyde formation as described previously [lo]. Oxygen concentrations were determined polarographically with a Clark electrode combined to an Eschweiler Combi-Analysator U, calibrated against commercial standard oxygen-nitrogen gas mixtures.

In a second series of experiments reactions were allowed to proceed in a closed vessel in the presence of an excess of bepzylamine (10 mM), and the time- course of the decrease in oxygen concentration was followed polarographically. Reaction velocities a t oxygen concentrations of 1.1,0.7, 0.5, 0.4 and 0.3 mM were determined by computer-programmed numeri- cal differentiation of the progress curves obtained using an initial oxygen concentration of about 1.2 mM (oxygen was passed through the reaction solution for 1 min immediately before reactions were started by the addition of enzyme), and velocities a t 0.25, 0.20 and 0.15 mM oxygen were determined similarly starting with air-saturated solutions. Pre- liminary experiments established that no inactiva- tion of the enzyme took place during the 10-20 min required for registration of progress curves, and that the small amounts of products accumulated during the reaction had no significant effect on the reaction velocity under the above conditions. Since benzylamine was used in “saturating” (though non- inhibitory) amounts, effects of benzylamine consumption may be neglected and the observations obtained at different oxygen concentrations can be attributed to a fixed benzylamine concentration of 10 mM.

For final estimation of kinetic parameters, observations from the two sets of experiments were pooled, appropriately weighted according to their individual variances, and submitted to iterative non-linear regression analysis by standard statistical methods described elsewhere [l 11.

Transient-State-Kinetic Methods Transient-state kinetic experiments were per-

formed in an Aminco-Morrow stopped-flow apparatus combined to a Beckman DU monochromator, an Aminco high-performance kinetic photometer, and a Kepco ABC-1000 high-voltage power supply for the photomultiplier tube. Photomultiplier signals over the time range 5 ms to 5 s were registered in a Tek- tronix 5103 N storage oscilloscope equipped with a 5A18N amplifier and a 5BlON time base. A Hewlett- Packard 7005B X-Y recorder was used to register signals over longer time periods.

Anaerobic conditions were attained by the pumping-flushing technique described by Carrico et al. [12]. Solutions being degassed and flushed with nitrogen were kept near 0 “C in anaerobic cells which were essentially identical with those described elsewhere [12].

Electron paramagnetic resonance spectra were recorded with a Varian E-3 spectrometer a t 77 K and about 9.2 GHz, using enzyme concentrations in the order of 0.15 mM. Signal intensities were measured by double integration of the spectra.

RESULTS Steady-State- Kinetic Experiments

Taylor et al., in a recent investigation of the steady state rate-behaviour of pig plasma benzylamine oxidase [5], have shown that linear and parallel Lineweaver-Burk graphs with respect to benzylamine are obtained a t different fixed concentrations of oxygen (and vice versa), i.e. that the enzymatic reaction between benzylamine and oxygen adheres to a rate-equation which may be written in the Dal- ziel form as

where v stands for the molar reaction velocity and S denotes benzylamine.

In order to obtain estimates of the Dalziel coeffi- cients q ~ i under the slightly different conditions used in the present investigation (pH 7.0 and 25 “C), determinations of the enzymatic reaction velocity were made for various oxygen concentrations (0.15 to 1.1 mM) a t a fixed concentration of benzylamine (10 mM), and for various benzylamine concentrations (0.1 - 10 mM) a t fixed oxygen concentrations of 0.25 mM and 1.25 mM. Lineweaver-Burk graphs with respect to benzylamine for the latter series of experiments (Fig. 1) were linear with identical slopes [confirming that Eqn (1) may be used for evahation of the data] from which a preliminary estimate of pz was obtained. Preliminary estimates of p1 and po were determined similarly from the slope and intercept, respectively, of the linear Lineweaver-Burk graphs with respect to oxygen obtained for the former

72

0.3

Kinetics of Benzylamine Oxidase

-

Eur. J. Riochem.

- 0.10.

E .- - 1 . c

0.05 -

0.15 1 / Fig. 3. Anaerobic reaction between benzylamine oxidase and substrate as fallowed by the increase in transmittance at 470 nm. Reaction solutions contained enzyme (11.8 pM) and benzylamine (A, 3.33 mM; B, 10.0 Mm) in 0.1 M phosphate buffer

pH 7.0

v /

OO 2 4 6 I / [O, ] (mM-')

Fig.2. Lineweaver-Burk graph with respect to oxygen at a fixed concentration of benzylamine (10 mM). Reactions were performed a t 25 "C in 0.1 M phosphate buffer pH 7.0 contain-

ing 1.01 pM benzylamine oxidase

series of experiments (Fig. 2 ) . Final estimates were computed statistically by iterative weighted fitting of Eqn (1) to the total number of observations indicated in Fig. 1 and 2, which gave yo = 0.9 ( 3-0.4) s, v1 = 1.5 (tO.25) s . mM and p2 = 1.0 (& 0.15) s . mM.

Anaerobic-Transient-State-Kinetic Experiments Benzylamine oxidase is pink and exhibits a

broad and fairly weak absorption in the visible region, with a maximum centered around 470 nm. When substrate is added under anaerobic conditions, there is a significant decrease in absorbance a t 470nm. This spectral change can be reversed by oxygenation of the solution [13].

Preliminary experiments established that the reaction between benzylamine oxidase and substrate was too fast to be followed by conventional spectro- photometric methods, and the stopped-flow technique was applied in order to study the time-dependence of the above absorbance changes a t 470 nm. Fig. 3 shows typical oscilloscope traces obtained on mixing the enzyme with benzylamine in the absence of oxygen, from which it can be seen that the rate of decolourization of the 470-nm chromophore, in contrast t o the amplitude of the absorbance change, is dependent upon the benzylamine concentration. The same final absorbance level was reached over a range of 0.05-10 mM benzylamine, and the approach to this level followed apparent first-order

Vol.35, Yo.1, 1973 A. LINDSTR~M, B. OLSSON, and G. PETTERSSON 73

kinetics, as indicated by the straight lines obtained in plots of log (AA,,,,) ws time (Fig.4). The apparent &&-order rate constants calculated from such plots were found to be proportional to the benzylamine concentration (Fig. 5), which suggests that the rapid decolourization of the 470-nm chromophore corresponds to a rate-determining bimolecular reaction between enzyme and substrate. The second-

, 0 1 2 3 4

T i m e ( s )

Fig.4. Semilogarithmic @oh of AA,,o v0 time for the anaerobic reaction between enzyme and benzylamine. Reaction solutions contained enzyme (9.7 pM) and benzylamine (A) 0.1 mM, (€5) 0.5 mM, (C) 1 mM, (D) 6 mM in 0.1 M phosphate buffer

pH 7.0

order rate constant for this process is indicated by the slope of the straight line in Fig.5; the value obtained by linear regression analysis of the data show in Fig.5 was 1.0 (f 0.1) s-l mM-l.

The copper-binding reagent cuprizone, which has been reported to inhibit benzylamine oxidase strongly [14], was found to have no effect on the 470-nm chromophoric changes when experiments were carried out by mixing the enzyme with a substrate solution containing the inhibitor (Fig.5). This might either indicate that binding of cuprizone to the enzyme does not affect the bimolecular reaction between enzyme and substrate, or that the interaction between enzyme and cuprizone is too slow t o have any significant kinetic effect during the short period of time required for reaction between enzyme and substrate. Steady-state kinetic experiments favoured the latter idea, in showing that preincubation of the enzyme with inhibitor was necessary to obtain any decrease in the initial rate of benzaldehyde formation. Since preincubation of benzylamine oxidase with 0.1 mM cuprizone was found to result in complete reduction of the 470-nm chromophore, the effect of the inhibitor on the anaerobic reaction (if any) between preincubated enzyme and benzylamine could not be studied apectrophotometrically.

Aerobic Transient-State Kinetic Experiments Fig.6 shows the time course of the change in

absorbance a t 470nm on mixing the enzyme with various amounts of benzylamine in the presence of 0.25 mM oxygen (air-saturated solutions). Absorb- ance changes have been given in two different time- scales, in order to indicate the presence of two distinct reaction phases. In the first phase, which shows an obvious correspondence to the fast decolouri-

[Benzylarnine] (mM )

Fig. 5 . Dependence on substrate concentration of apparent first- order rate constants for dewlmrization of the 470-nm chromophore. (0). Data obtained for the interaction between benzylamine oxidase (about 10 pM) and substrate under anaerobic conditions: (+) corresponding data obtained in the presence

of 0.25 mM oxygen (air-saturated solutions) ; (0) observations obtained anaerobically by mixing the enzyme with benzylamine solutions containing 0.1 mM cuprizone. Each observation represents a separate experiment and gives the mean of

3-5 shots in the stopped-flow apparatus

74 Kinetics of Benzylamine Oxidase Eur. J. Biochem.

0.04

E 3 0

c m a c 40.03 0 Lo

9

0 2 4 0 100 200 300 400 500 Time ( s )

Fig. 6. Time-course of 470-nm chrornophoric changes on mizing benzylamine oxidme with substrate .under aerobic conditions. Reaction solutions contained enzyme (9.8 pM) and benzylamine (A) 0.1 mM, (B) 0.5 mM, (C) 5 mM, m 0.1 M phosphate buffer pH 7.0, equilibrated with air (0.25 mM oxygen). (0) Theoretical progress curves calculated under the assumption that steady-

state conditions prevail after the initial rapid reaction phase

zation process observed anaerobically, there is a rapid decrease in absorbance towards a steady-state level which is reached within a few seconds. When the initial benzylamine concentration exceeds 0.25 mM, this fast phase is followed by a slow further decrease in absorbance with half-times in the order 3-5 min, yielding a h a 1 absorbance corresponding to that obtained after compIeted reaction under anaerobic conditions. At substrate concentrations lower than 0.25 mM the initial rapid phase is followed by a slow increase in 470-nm absorbance, resulting in complete recolourization of the reaction solution after 5-15 min.

Apparent first-order rate constants calculated for the approach to the steady-state level of absorbance were found to be about 1 s-1 larger than those obtained anaerobically, but showed a similar linear dependence on the benzylamine concentration (Fig. 6). Consequently, the fast chromophoric changes observed aerobically and anaerobically appear to be due t.0 the same bimolecular reaction between enzyme and substrate, the rate of which is unaffected by the presence of oxygen. The intercept effect of about 1 s-l obtained in the presence of oxygen is of the same order of magnitude as l/~l,,, and can be attributed to cycling of the enzyme under aerobic conditions.

As can be seen from Fig.6, there is only a partial reduction of the 470-nm chromophore a t the end of the fast reaction phase under aerobic conditions, corresponding to about 60°/, decrease in absorbance (measured with reference to the total absorbance change AA, observed under anaerobic conditions) a t substrate concentrations in the order of the Km-value for benzylamine in air-saturated solutions (see Fig. 1). This observation suggests that the steady- state level of 470-nm absorbance reflects the steady-

0

1 I I I 5 10 15 20

l / [Benzylamine] (mM-')

Fig. 7. Double-reciprocal plot of the steady-state level of 470-nm absorbance at different bemylamine concentrations. Relative absorbance changes (dA/dA,) were recorded at the end of the fast reaction phase in experiments similar to and including

those shown in Fig. 6

state proportion of free enzyme, which for any first- degree Wong-Hanes mechanism in which free enzyme only participates in a bimolecular reaction with substrate is given by [l + ([S]/Km)]-l [15]. If such is the case, the absorbance change AA at the end of the fast reaction phase would be given by

AA [Sl/(Km + [Sl). (2) Confirmatively, experimental observations of the quotient AAIA, fall well along a straight line in the double-reciprocal plot shown in Fig.7. The slope of the line, calculated by weighted regression analysis,

Vol. 35, No. 1,1973 A. LINDSTROM, B. OLssa

corresponds to K , = 0.17 mM, which agrees well with the K , of 0.15 mM estimated from the steady- state kinetic data in Fig. 1.

I n view of this result, the absorbance changes observed during the slow reaction phase can be interpreted as a readjustment of the steady-state proportion of free enzyme. The reaction catalyzed by benzylamine oxidase is known to proceed under consumption of equimolar amounts of oxygen and benzylamine. When the latter substrate is present in excess to oxygen, substrate consumption will lead to a gradual approach anaerobic conditions, and the 470-nm absorbance wil l decrease to the h a 1 level obtained in the absence of oxygen. When oxygen is present in excess to benzylamine, substrate consumption will favour reoxidation of the enzyme and yield a gradual increase in 470-nm absorbance.

Quantitative evidence in favour of this inter- pretation of the slow phase reactions was obtained by calculation of theoretical progress curves using the Dalziel coefficient estimates given above. With Dalziel notations Eqn (2) takes the form

d A = dAC0 * (Po + P1/[021)/ (Po + 97,/ro21 + PZ/[Sl). (3)

Observing that the reaction velocity v in Eqn (1) is identical with -d[S]/dt and -d[02]/dt, integration of Eqn (1) yields

Po (cs - PI) + 91 1n (COa h 0 2 1 ) + Pz In (cS/[sI) = CE . t (4) where cs, coS and CE denote initial concentrations of benzylamine, oxygen and enzyme, respectively. Eqns (3) and (4) may be used to calculate joint values of d A and t a t different levels of substrate concentrations under the assumption that steady-state conditions prevail. Theoretical progress curves calculated for the experiments given in Fig.6 are indicated by open circles in this figure, and show a most satis- factory qualitative and quantitative consistence with the experimental results.

Optical- Absorption Spectra The anaerobic reaction between enzyme and

benzylamine was followed by the stopped-flow technique a t different wavelengths within the range 330-600 nm. Consistent estimates of the apparent first-order rate constant for decolourization of the enzyme were obtained a t all wavelengths studied, which indicates that only one reaction yielding chromophoric changes in the visible region is involved. Confirmatively, difference spectra with reference to the initial absorbance of the reaction solution showed the same shape at any time during the reaction, and agreed with the difference spectrum obtained after completed reaction. As shown in Fig. 8, this spectrum consists of a singIe line centered around 470 nm (AA,,,, = 1600 M-l cm-I).

IN, and G. PETTERSSON 75

a.

O * O t a

a.

O e 0 i a

t

," 0.01 i a o o o o o o * a

400 500 600 0

Wavelength (nm)

Fig. 8. Absorbance-difference spectrum for the anaerobic reaction between benzylamine oxidase and substrate. Absorbance changes indicated were measured 0.5 s (0) and 5 s ( 0 ) after mixing 25 pM enzyme with an equal volume of 2 mM sub-

strate in 0.1 M phosphate buffer pH 7.0

Electron- Paramagnetic- Resonance Spectra Fig. 9 shows low-temperature electron para-

magnetic resonance spectra of benzylamine oxidase before and after mixing with an excess of substrate under anaerobic conditions. The small substrate- induced changes in the shape of the signal have previously been interpreted as being due exclusively to g-value shifts [8], but a significant decrease in the magnitude of the hyperfine splitting constant also appears to be involved. Anyhow, these spectral changes could be observed in reaction solutions frozen immediately after decolourization of the 470-nm chromophore (within I0 s after mixing), and no further spectral changes were obtained by allowing reaction solutions to stand a t room-temperature for 5-20min before spectra were recorded. Double-integration of the spectra established that no significant reduction of copper took place even after a reaction time of 20 min.

Similar results were obtained under aerobic conditions using 150pM enzyme and 0.5mM or 5 mM substrate. Electron paramagnetic resonance spectra recorded a t the end of the fast reaction phase (within 1 0 s after mixing), or during a steady-state phase of the reaction corresponding to 80-1000/0 reduction of the 470-nm chromophore, were essentially identical with the one in Fig.9B. whereas spectra recorded after recolourization of the 470-nm chromophore were indistinguishable from that of native

76 Kinetics of Benzylamine Oxidase Eur. J. Biochem.

I I I V A 2600 2800 3000 3200

Magnet ic f ie ld (gauss )

Fig. 9. Electron-paramagnetic-resonance spectra of benzylamine oxidase at 77 K and 9.21 GHz. Spectra were recorded after mixing of 0.4 ml of 186 pM enzyme under anaerobic conditions with 0.1 ml of (A) phosphate buffer pH 7.0 and (B) phosphate buffer pH 7.0 containing 26 mM benzylamine.

Reaction solutions were frozen within 10 s after mixing

enzyme. Signal intensities of spectra recorded at different reaction stages did not show any significant variation.

DISCUSSION Pig plasma benzylamine oxidase and pig kidney

diamine oxidase are closely related in several re- spects [7,13,14]. Both enzymes contain copper and pyridoxal phosphate, and catalyze the oxidation of various amines by molecular oxygen under formation of the corresponding aldehydes, ammonia and hydro- gen peroxide. Absorption spectra of the enzymes are similar, as are the chromophoric changes on addition of substrates and inhibitory carbonyl reagents. Steady-state kinetically, both enzymes conform to a rate equation of the Dalziel type with an insignificant magnitude of the y12-term, which has been taken as evidence for a ping-pong type of mechanism [3,5]; the mechanism suggested for benzylamine oxidase on the basis of product-inhibition studies [5] is shown in Scheme 1. Some differences in the transient-state kinetic behaviour of the two enzymes are, however, evident in view of the present results.

Mondovi et al. [9] found that addition of substrate to pig kidney diamine oxidase under anaerobic conditions resulted in a biphasic decrease in absorbance at 500 nm. The fast reaction phase was suggested t o reflect the formation of an enzyme - substrate complex, whereas the slow phase was found to cor-

Scheme 1. The ping-pong mechanism suggested by Taylor etal. [5] for reactions catalyzed by benzylamine oxailme. S stands for monoamines functioning as substrates and P for the corresponding aldehydes formed as products. Xr denote

intermediately formed enzymatic species

respond to a reduction of the enzyme, more specifical- ly to a reduction of enzyme-bound copper. With benzylamine oxidase, however, only one single phase of rapid decolourization of the corresponding chromophore can be observed after addition of substrate under anaerobic conditions (Fig. 3). Biphasic progress curves were obtained in the presence of oxygen, but the slow chromophoric changes observed under such conditions can be entirely attributed to a readjustment of the steady-state level of free enzyme, caused by substrate and oxygen consumption during the reaction.

The initial rapid decrease in 470-nm absorbance on addition of substrate to benzylamine oxidase undoubtedly reflects a rate-limiting formation of a binary enzyme - substrate complex, the rate of which process appears to be unaffected by the presence of oxygen. From a kinetic point of view, decolourization of the 470-nm chromophore may be considered to occur simultaneously with substrate- binding to the enzyme. If complex formation takes place prior t o reduction of the 470-nm chromophore, the subsequent reactions leading to chromophoric changes must be very rapid in comparison to the substrate-binding step, as evidenced by the absence of a saturation effect on the apparent first-order rate constant a t high benzylamine concentrations.

The present results indicate that the steady-state kinetic coefficient ya equals the reciprocal value of the second-order rate constant (k, in Scheme 1) for the binding of substrate to the enzyme. When interpreted in view of the mechanism shown in Scheme 1, for which we have yz = (1 + k-l/kz)/kl, this observation provides evidence that dissociation of substrate from the enzyme is much slower than the forward isomerization or breakdown of the enzyme - substrate complex (k1 < k2), i .e . that binding of substrate to the enzyme kinetically may be considered as irreversible. Confirmatory evidence for an essentially irreversible binding of substrate to benzylamine oxidase ia given by the observation that complete reduction of the 470-nm chromophore is obtained anaerobically at the lowest substrate concentrations tested, also, which establishes that the equilibrium concentration of free enzyme under such conditions (10 pM enzyme and 50 pM substrate) is negligibly small.

Vo1.35, No.1, 1973 A. LINDSTRON, B. OLSSON, and G. PETTERSSON 77

Examination of Fig. 2 shows that the maximum activity of benzylamine oxidase under physiological conditions is mainly controlled by the oxygen concentration. Even though the presence of one or several rate-limiting substrate- and oxygen-independent reaction steps in the catalytic mechanism is evidenced by the significance of the qo-term in the Dalziel rate equation, velocity constants for such reaction steps must equal or be larger than l/yo (about 1 s-l), which should be compared with the maximum activity of 0.15 s-1 obtained in air-saturated solutions. The observation that l/qo and the intercept deviation of apparent first-order rate constants for aerobic reduction of the 470-nm chromophore (Fig.5) are of closely agreeing magnitude strongly suggests that only one single rate-limiting substrate- and oxygen-independent reaction step, with a velocity constant in the order of 1 s-l, is kinetically significant.

A valence change of copper could not be detected a t any stage of the reaction between benzylamine oxidase and substrate, irrespectively of whether or not oxygen was present in the reaction solution. This does not exclude that reduction of copper may be an obligatory in the catalytic mechanism; as pointed out by Mondovi et al. [9], a rapid redox equilibration between certain enzymatic species, favouring those containing copper in the oxidized state, would result in a less than stoichiometric reduction of the electron paramagnetic resonance signal by substrate under anaerobic conditions. If such a situation is at hand in the case of benzylamine oxidase, it may be con- cluded that steady-state and anaerobic equilibrium concentrations of enzyme species containing copper in the reduced state are less than about 5 O / , of the total enzyme concentration. The present investigation does not, however, give any support for a valence change of copper during catalysis. The results obtained are fully consistent with previous suggestions that amine oxidases dependent on copper and pyri-

doxal phosphate operate by mechanisms in which copper remains bivalent during catalysis [6 - 81.

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

REFERENCES 1. Kapeller-Adler, R. (1970) Amine Oxidases and Methods

2. Yamasaki, E. F., Swindell, R. & Reed, D. J. (1970) Bio-

3. Finazzi Agrb, A., Rotilio, G., Costa, M. T. & Mondovi, B.

4. Oi, S.. Inamasu. M. & Yasunobu. K. T. (1970) Biochem-

for Their Study, pp. 26-28, Wiley, London.

chemistry, 9, 1206-1210.

(1969) FEB8 Lett. 4, 31-32. \ I

istry, 9, 337813383. 5. Tavlor, C. E.. Tavlor, R. S.. Rasmussen, C. t Knowles.

@. F: (1972) Biochek. J . i30 , 713-728. 6. Yamada, H. & Yasunobu, I(. T. (1962) J . Biol. Chem.

7. Mondovi, B., Costa, M. T., Pinazzi Agrb, A. & Rotilio, G. (1968) in Pyridoxal Catalysis : Enzymes and Model Systems (Snell, E. E., Braunstein, A. E., Severin, E. S. & Torchinsky, Y. M., eds) pp. 403-423, Inter- science, New York.

8. Buffoni, F., Della Corte, L. & Knowles, P. F. (1968) Biochem. J . 106, 575-576.

9. Mondovi, B., Rotilio, G., Finazzi Agrb, A., Vallogini, M. P., Malmstrom, B. G. & Antonini, E. (1969) FEBS Lett. 2, 182-184.

237, 3077-3082.

10. Lindstrom, A. & Pettersson, G. (1973) Eur. J . Biochem.

11. Pettersson, G. & Pettersson, I. (1970) Acta Chem. Scand.

12. Carrico. R. J., Malmstrom. B. G. & Viinn&,rd. T. (1971)

34, 564-568.

24, 1275-1286. " I \ I

Eur..J. Biochem. 20, 518-524. 13. Buffoni, F. & Blaschko, H. (1964) Proc. R. Soc. Lond. B

14. Blaschko, H. & Buffoni, F. (1965) Proc. R. Soc. Lond. B

15. Wong, J . T. F. & Hams, C. S. (1962) Can. J . Biochem.

BWl. Sci. 161, 163-167.

Bi01. Sci. 163, 45-60.

Physiol. 40,763-789.

A. Lindstrom, B. Olsson, and G. Pettersson Avdelningen for Biokemi 1, Kemicentrum, Lunds Universitet Box 740,s-220 07 Lund 7, Sweden

Documents

Kinetics of the Interaction between Pig-Plasma Benzylamine Oxidase and Substrate