The Reaction Pathway of Pig Brain Mitochondria1 Monoamine Oxidase

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1 European J. iochem. 5 (1968) The Reaction Pathway of Pig rain Mitochondria1 Monoamine Oxidase K. F. TIPTON Department of iochemistry, University of Cambridge (Received March 21, 1968) Initial rate measurements of the oxidation of tyramine were carried out with purified pig brain mitochondrial monoamine oxidase. The reciprocal plots obtained when the concentration of either substrate was varied at a series of fixed concentrations of the other gave families of parallel lines consistent with a kinetic mechanism in which a ternary complex is not involved. This mechanism was supported by product inhibition studies and the demonstration of a half-reaction in the absence of oxygen. The mitochondrial enzyme monoamine oxidase catalyzes the reaction RCH,NH, + 0, + H,O +.+ RCHO + NH, + H,O,. There have been a number of reports that the activity of monoamine oxidase increases when the oxygen tension in the assay medium is increased [l-31. In this study the kinetics of tyramine oxidation were investigated at a series of different oxygen concentrations using a purified preparation of monoamine oxidase from pig brain mitochondria. preliminary account of part of this work has been reported [4]. MTERILS ND METHODS Monoamine oxidase was purified from pig brain mitochondria by the method previously described [5]. The activity of monoamine oxidase was assayed using a Clark oxygen electrode connected through a voltage divider to a Honeywell-rown 1 mv strip-chart recorder, the apparatus being similar to that described by Dixon and Kleppe [6]. The oxygen uptake was determined by the method of Creasey[7] and the reaction mixture contained, in a total volume of 2.4 ml: 200 pmoles of sodium phosphate buffer (ph 7.0), 100 units of catalase, 20 pmoles of semicarbazide, 2pmoles of KCN, and tyramine. The reaction wafs started by the addition of enzyme. Under these conditions each mole of tyramine oxidized caused the uptake of 1 p atom of oxygen. Catalase was omitted in the experiments where hydrogen peroxide was used as an inhibitor and semicarbazide was omitted when p-hydroxybenz- Enzymes. Monoamine oxidase or monoamine: 0, oxidoreductase (deaminating) (EC ) ; alcohol dehydrogenasr or alcohol: ND oxidoreductase (EC ); L-aminoacid oxidase or L-aminoacid: 0, oxidoreductase (deaminating) (EC ) ; L-aspartate: 2-oxoglut,arate aminotransferase (EC ); succinic dehydrogenase or succinate: (acceptor) oxidoreductase (EC ). aldehyde was used. ll assays were performed at 30" and the specific activity is expressed asp atoms oxygen consumed per mg protein per minute. The oxygen concentration in the assay medium was varied by gassing with oxygen using the method described by Dixon and Kleppe [6]. ldehyde production was assayed by following the decrease in the fluorescence of NDH as it was oxidized by the aldehyde in the presence of yeast alcohol dehydrogenase. The reaction mixture contained, in a volume of 1.O ml : 90 pmoles of phosphate buffer ph 7.6, 0.05 mg of NDH, 1.25 pmoles of tyramine, and 30 units of yeast alcohol dehydrogenase. Fluorescence measurements were made at 30" using an minco-owman spectrophotofluorometer coupled to a ryansx-y recorder, with 340 mp as the excitation wavelength and 460 mp as the emission wavelength. Samples of the assay medium were depleted in oxygen by bubbling nitrogen through them in a Nilox deoxygenator, and assays were carried out in an atmosphere of nitrogen. calibration curve was constructed using samples of a standard acetaldehyde solution. Protein concentrations were estimated by the microbiuret method [8], using bovine serum albumin as a standard. Measurements of absorbance were carried out using a Unicam SP 500 spectrophotometer with quartz-glass cuvettes of 1 cm light path, and measurements of ph were made using a Radiometer PHM 22r ph meter. ovine serum albumin was obtained from rmour Chemical Co. Ltd; catalase, NDH and yeast alcohol dehydrogenase from iochemica oehringer (Mannheim, Germany) and oxygen and nitrogen from ritish Oxygen Co. Ltd. ll other chemicals were obtained from ritish Drug Houses Ltd or Hopkin and Williams Ltd, and were of the highest quality available. Distilled water was passed through a Permutit Mark I1 deionizer before use.

2 Vol.6, No.3, 1968 K. F. TIPTON 317 RESULTS The K, value for tyramine oxidation by purified pig brain monoamine oxidase, using air saturated buffer, has been previously reported [5]. Fig. 1 shows the reciprocal plots [9] obtained when the initial rates of tyramine oxidation were determined at varying tyramine concentrations and a series of fixed oxygen concentrations; a series of parallel lines are obtained. Similar parallel reciprocal plots were obtained when the oxygen concentration was varied at a series of fixed tyramine concentrations (Fig. 1 ). Reciprocal plots in which the slopes are unchanged, rega,rdless of the concentration of the second substrate, are consistent with a mechanism in which the reaction proceeds through a modified form of the enzyme and a series of binary complexes, without the formation of kinetically significant amounts of a ternary complex. Such a mechanism has been termed a ping-pong mechanism by Cleland [lo]. Scheme 1 show shows a possible mechanism of this type : E E Scheme 1 Steady state treatment of this mechanism yields an equation of the form: l/ctyrminei (mm-') 2 4 1/C021 (mm-') Fig.1. The kinetics of oxidution of tyramine by rnonournine oxidase. () Reciprocal plots of initial velocities against tyramine concentration at a series of fixed oxygen concentrations. Oxygen concentration: H, 0.93 mm;, 0.79 mm; 0, 0.65 mm and 0, 0.23 mm. () Reciprocal plots of initial velocities against oxygen concentration at a series of fixed tyramine concentrations. Tyramine concentration :, 2.5 mm; 0, 1.OmM; 0, 0.5mM, and, 0.25mM [El represents the total enzyme concentration, V represents the velocity when substrate and oxygen concentrations are infinite, K& is the Michaelis constant for substrate at infinite oxygen concentration and K: is the Michaelis constant for oxygen at infinite substrate concentration. Values for K& and K; were determined from secondary plots of the kinetic data be 240 pm and 234 pm respectively. Evidence in support of the proposed mechanism was obtained from the pattern of inhibition given by the products. It follows from scheme 1 that if the aldehyde product is present, and the initial combination of E with product is the only kinetically significant step that takes place between the two, the rate equation becomes : V Scheme 2

3 318 Reaction Pathway of Monoamine Oxidase Xiiropean J. iochem. where Kb is the dissociation constant of the E'P complex and hence represents the inhibitor constant for the product acting as an inhibitor. From this equation it can be seen that P should behave kinetically as a competitive inhibitor with respect to 0, and an uncompetitive inhibitor with respect to substrate. Fig.2 shows that the inhibition pattern obtained when p-hydroxybenzaldehyde was used to represent the aldehyde products is consistent with this formulation. In the case of monoamine oxidase, it may be the inhibition by hydrogen peroxide could, however, be complicated by the fact that it has been found to be an irreversible inhibitor of a number of enzymes. In the case of monoamine oxidase, it was found that when the enzyme was incubated with 1.O mm H,O, at 30", the extent of inhibition did not increase with time. It has been reported that irreversible inhibition of diamine oxidase by hydrogen peroxide requires the presence of substrate [ill, and in the case of chymotrypsin, it has been shown that irreversible inhibition by hydrogen peroxide is more rapid in the presence of substrate [12]. To investigate whether this was the case with monoamine oxidase, the experiment shown in Fig.3 was carried out. The addition of catalase to the enzyme-substrate-per- L I I I I I l/ityrminei (mm-') 1.0 t Pig. 3. The reversible inhibition of monoamine oxidase by hydrogen peroxide. Details of the assay medium are as given in the text with catalase omitted from the mixture. t point enzyme was added, at point 100 pl of 0.25 M hydrogen peroxide was added and at point C 100 units of catalase were added and the oxygen electrode was removed to allow some of the evolved oxygen to escape. The electrode was then replaced and the oxygen Concentration and the rate of oxygen consumption were determined /[021 (mm-') Fig.2. The inhibition of monoamine oxidase by p-hydroxybenzaldehyde. () Reciprocal plots of initial velocities against tyramine concentration at different inhibitor concentrations. The oxygen concentration was 0.23 mm and the inhibitor concentrations were 40yM (0) or 20pM () or zero (0). () Reciprocal plots of initial velocities against oxygen concentration at different inhibitor concentrations. The tyramine concentration was 2.5 mm and the inhibitor concentrations were 100 pm (0) or 50 pm () or zero (0) expected that by analogy with the amino-acid oxidases, the initial product may not be an aldehyde, but an imine, which is then spontaneously hydrolysed to the aldehyde. If this were the case, the aldehyde used would be acting as a product analogue rather than the product itself. y a similar mechanism to that shown inscheme2, the second product H,O, would be expected to be a competitive inhibitor with respect to tyramine, and uncompetitive with respect to oxygen. nalysis of oxide mixture would be expected to reduce the measured reaction velocity by one half, but since the oxygen concentration has been increased by the decomposition of the hydrogen peroxide, the velocity obtained should be greater than this. The final velocity obtained from Fig. 3 was 2.15 p, atoms O,/niin, compared with an expected value of 2.18 p atoms O,/min calculated from a Michaelis curve of the variation of enzyme activity with 0, concentration. Fig.4 shows the inhibition pattern obtained with hydrogen peroxide as a product inhibitor. The inhibition pattern obtained can be seen to differ from the competitive-uncompetitive pattern to be expected from the simple treatment in Scheme 2. Inhibition is mixed with respect to both 0, and tyramine. mixed-mixed type of inhibition pattern obtained with respect to the two substrates could be explained on a scheme in which H,O, is not only bound to the normal form ofthe enzyme (E), but also the modified form (E'). This would give rise to a kinetic equation of the type : V Scheme 3

4 Vol.5, No.3, 1968 K. F. TIPTON TYRM I NEI (mm- ) duced during the reaction implies that it should be possible to demonstrate the occurrence of the half reaction leading to the production of the modified enzyme E + S + ES + E P + E + Pi in the absence of oxygen. In order to observe this half reaction, the aldehyde produced was coupled to the oxidation ofndh using yeast alcohol dehydrogenase. The burst of product released when enzyme was added to the reaction mixture is shown in Fig. 5. In this experiment it was not 0 1/[021 (mm ) Fig.4. The inhibition of monoamine oxidase by hydrogen peroxide. () Reciprocal plots of initial velocities against tyramine concentration at different inhibitor concentrations. The oxygen concentration was 0.23 mm and the inhibitor concentrations were mm (0) mm () or zero (0). () Reciprocal plots of initial velocities against oxygen concentration at different inhibitor concentrations. The tyramine concentration was 2.5 mm and the inhibitor concentrations were 0.77 mm () 2.3 mm (m) or zero (0) Table. The inhibition of monoamine oxidase by products Kinetic definitions of the inhibitor constants are given in the text. The inhibitor constants were calculated from the slopes of the reciprocal plots Inhibitor Substrate p-hydroxy- Hydrogen peroxide benzsldehyde Ki & K; (LM wan (L1 Tyramine Oxygen a Calculated assuming a value for V of 5.1 (r atoms O,/mg/min. where k i and K; are the dissociation constants of the E.H,O, and E H,O, complexes respectively. The Kg values for the products acting as inhibitors are shown in the Table. The proposed mechanism in which a modified form of the enzyme but no ternary complex is pro- I I I I TIME (sec) E ig.5. The formation of aldehyde by monoamine oxidase in a medium depleted of oxygen. ldehyde was assayed by following the decrease in fluorescence of NDH as it was oxidised by the aldehyde in the presence of yeast alcohol dehydrogenase. t point, 100 p1 of enzyme (0.209 mg/ml) were added. t point, 25 pl of enzyme were added and at point C, 25 pl of M tyramine were added possible to reduce the oxygen concentration in the assay medium to zero, and so a final rate of turnover of the modified enzyme was obtained after the initial burst. However, by extrapolating this final rate to zero time, the aldehyde which would be produced by the conversion of all the native enzyme to the modified form can be calculated as shown in Fig.5. The active centre weight of the enzyme calculated from this burst was 111,000, close to the molecular weight of the enzyme estimated by gel-filtration [5], although one would expect the relatively high turnover rate of E to came an apparent overestimation of th+ molecular weight. DISCUSSION The kinetic studies in this paper are in accordance with a mechanism in which a modified form of the enzyme is produced with no kinetically significant formation of a ternary complex, and the inhibitions by products are consistent with this mechanism. The formation of a modified form of the enzyme during

5 320 K. F. TIPTON: Reaction Pathway of Monoamine Oxidase European J. iochem. the reaction is in agreement with studies which indicate that the flavine component of the enzyme becomes either fully or partially reduced in the presence of substrate [13]. The kinetic measurements do not indicate whether the modified enzyme represents a reduced or a partially reduced form of the enzyme. Massey and Curti [14] have pointed out that the appearance of parallel lines in the Lineweaver-urk plots is not necessarily diagnostic of a mechanism which does not involve a ternary complex; L-amino acid oxidase, which gives such Lineweaver-urk plots, has been shown to form a ternary complex during its reaction, and they point out that suitable values for the kinetic rate constants would give rise to parallel lines kinetics, despite the involvement of a ternary complex in its mechanism. similar finding has been reported for alcohol dehydrogenase by Dalziel [ 151. However, the pattern of inhibition obtained with products and the observation of a burst of aldehyde production at low oxygen concentrations are difficult to reconcile to a mechanism involving a kinetically significant ternary complex. Qawron et at. [16] have shown that the kinetic mechanism of succinic dehydrogenase changes on solublization of the enzyme, and Jenkins and D ri [ 171 have shown that the ping-pong kinetic mechanism of L-aspartate : 2-oxoglutarate amino transferase is dependent on the composition of the assay medium used. These results indicate that a kinetic reaction pathway cannot be regarded as an immutable mechanism, but will depend on the conditions of the assay in that these may affect the relative values of the individual kinetic rate constants. The skilled technical assistant of Mr. I. P. C. Spires is gratefully acknowledged. REFERENCES 1. Philpot, F. J., iochem. J. 31 (1937) Kohn, H. I., iochem. J. 31 (1937) Novick, W. J., iochem. Pharmaeol. 15 (1966) Tipton, K. F., bstracts 4th FES Meeting (Oslo), 1967, p Tipton, K. F., European J. iochem. 4 (1968) Dixon, M., and Kleppe, K., iochim. iophys. ctu, 96 (1965) Creasey, N. H., iochem. J. 64 (1956) Goa, J., J. Glin. Lab. Inwest. 5 (1953) Lineweaver, H., and urk, D., J. m. Ghem. SOC. 56 (1934) Cleland, W. W., iochim. iophys. cta, 67 (1963) Mondovi,., Rotilio, G., Finazzi-grb,., and Costa, M. T., iochim. iophys. cta, 132 (1967) Dixon, G. H., and Schachter, H., Cunad. J. iochem. 42 (1964) Tipton, K. F., iochem. J. 104 (1967) 36P. 14. Massey, V., and Curti,., J. iol. Chem. 242 (1967) Da,lziel, K., iochem. J. 84 (1962) Gawron, O., Mahajan, K. P., Limetti, M., Kananen, G., and Glaid,. J., iochemistry, 5 (1966) Jenkins, W. T., and D ri, L., J. iol. Chern. 241 (1966) K. F. Tipton Department of iochemistry, The University Tennis Court Road, Cambridge, England