Regulatory Properties of a Plant NAD: Isocitrate Dehydrogenase

Transcription

1 European J. Riochem. 7 (1969) Regulatory Properties of a Plant NAD: Isocitrate Dehydrogenase The Effect of Inorganic Ions T. P. COULTATE and D. T. DENNIS Unilever Research Laboratory, Colworth House Sharnbrook, Bedford (Received July 17, 1968) 1. Some properties of NAD : isocitrate dehydrogenase from swede (Brassica napw L.) are described. This enzyme shows simpler regulatory properties than that from animals or fungi. 2. Isocitrate, citrate and tricarballylate are positive effectors but neither ADP nor AMP have any effect. Pyruvate, acetate, aketoglutarate, succinate and DLmalate do not stimulate or inhibit at 2 mm. 3. ATP and other nucleoside triphosphates inhibit this enzyme but the inhibition appears to be due to the formation of a complex with the activating cation. 4. The principal control of the enzyme is a potent inhibition by NADH competitive with NAD. Elevation of either isocitrate or activating cation concentration lowers the Km for NAD and in this respect MnSO, is more effective than MgSO,. In contrast, the Ka for NADH is.5 mil with 1 mm MgSO, and.16 mm with 1 mm MnSO,. 5. The enzyme is inhibited by high concentrations (> 2 d) of inorganic anions. This inhibition is apparently competitive with isocitrate but anions most likely cause a conformational change. 6. The interactions between isocitrate and activating cation have been examined. Elevation of the concentration of either isocitrate or cation raises the affinity of the enzyme for the other to a limiting value. The nature of the cation has only a slight effect on the minimum Km for isocitrate (.35 mm with MgSO,,.25 mm. with MnSO,) but the minimum value of the K, for MgSO, is 1 p,m compared with 7 pm for MnSO,. 7. It is concluded that the enzyme binds one molecule each of isocitrate, activating cation and NAD or NADH at the catalytic site and one molecule of citrate or isocitrate at an effector site. A preliminary report [l] has described some properties of the NAD : isocitrate dehydrogenase from swede (Bramica; m pl) mitochondria. The enzyme is very unstable and shows cold lability. In common with the enzyme from other sources it is activated by citrate and inhibited by NADH but in contrast the activation by AMP or ADP is not observed. NAD : isocitrate dehydrogenase from pea mitochondria has very similar properties [Z]. The enzymes from Neurospora [351 and yeast [6,7] are activated by citrate, AMP and NAD. Magnesium ions also activate the yeast enzyme [6]. NADH inhibits the Neurospora [3] and mammalian heart [9] enzymes competitively but it has not been examined in yeast. In animals ADP and not AMP is the activator [S,Q]. Unusual Abbreviations. 2(NZhydroxyethylpiperazin N'y1)ethanesulphonic acid, Hepes ; Ntris (hydroxymethyl) methyl2aminoethanesulphonic acid, Tes. 3nzyrne.s. NAD : isocitrate dehydrogenase, threod. isocitrate : NAD oxidoreductase (EC ); glutamate dehydrogenase, Lglutamate : NAI) oxidoreductase (deaminating) (EC ); phosphofructokinase, ATP : DfmCtosetiphosphate 1phoaphotransferase (EC ); malate dehydrogenase, r,malate : NAD oxidoreductase (EC ). 11 European J. Biochem., Vol. 7 The enzyme from yeast is inhibited by inorganic anions [lo] and this mhibition is reversed by isocitrate [lo], AMP and Mg++ [ll, 121. Cox and Davies [Z] examined the effects of inorganic anions on the pea enzyme by the use of Hepes buffer which allowed the anion concentration to be reduced to less than 1 mm. They demonstrated increasing inhibition in the series F, C1, Br and I although the plots of substrate concentration against rate remained sigmoid even in the absence of anions. With the exception of the study of the mammalian heart enzyme by Chen and Plaut 19,131 magnesium ions rather than manganese ions have been used as cofactor. The effectiveness of various cations as cofactors for the pea seedling [23 and yeast enzymes [6,1,14] has been studied. Both these enzymes, and the enzyme from bovine heart [13], show a much greater afsnity for Mi++ than Mg++. It is notable, therefore, that Mg++ is usually regarded as the factor of choice. Cox and Davies [2] found a slight inhibition by elevated concentrations of Zn++. We demonstrate in this report the importance of inorganic ions to the activity of isocitrate dehydrogenase. In particular the effectiveness of manganese

2 154 Regulation of a Plant NAD: Isocitrate Dehydrogonase European J. Biochem. and magnesium as cofactors is contrasted and the influence of inorganic anions is confirmed. METHODS Preparation of Enzyme The procedure for the isolation of the enzyme from swede storage tissue was described in the earlier report [l]. The buffer used for the isolation of the mitochondria differed in that magnesium sulphate was omitted and 2. mm EDTA included. A yield of about 75 mg of acetone powder per kg of swede was obtained. No appreciable loss of activity of the powder was detected after storage for one month at 2". Immediately prior to use the acetone powder was suspended in 5 mm Tes buffer ph 7.4 containing 5 M glycerol using a polytetrafluoroethylene homogenizer. 5M glycerol has been shown to stabilize the enzyme [2]. A concentration of 1 mg acetone powder per ml usually gave sufficient activity. The suspension was centrifuged at 2, x g for 5min and supernatant stored at " till required. Under these condit,ions the preparation was stable for up to 2 hours. Assay The standard assay contained 1.O mm magnesium sulphate, 1. mm NAD and 1. mm misocitrate brought to a volume of 2.4 ml at ph 7.4 with 5 mm TesNaOH buffer. The reaction was initiated by the addition of.1 ml of enzyme preparation. The course of the reaction was followed by measuring the increase in absorbance at 34 mp in 1 cm cuvettes using a Unicam SP 8 spectrophotometer fitted with an external recorder. The cell compartment of the spectrophotometer was thermostated to 25". Concentrations of isocitrate cited are the concentrations of the racemic mixture. Deviations from the standard assay are indicated in the relevant sections. Biochemical reagents were purchased from Sigma (London) except for tricarballylic acid which was purchased from British Drug Houses. Specific Activity In a completely saturating reaction mixture (1 mm isocitrate, 2 mm NAD and 1 mm MnSO,).1 ml of a typical enzyme preparation reduced 7.3 nmoles of NAD per minute. Determination of the protein content of the preparation by the Lowry method [15] (with bovine serum albumin as standard) indicated a specific activity of.23 EC units per mg of protein. When the enzyme preparation was centrifuged at 5, xg or 1 min the activity was reduced to 6.6 nmoles of NAD per minute per mg of acetone powder but the specific activity was raised to.59 EC units per mg protein. RESULTS Effect of ph The reaction rate as a function of ph is shown in Fig.1. It will be seen that neither elevated Mg++ levels, replacement of Mg++ with Mn nor the presence of AMP affect the position of the ph optimum or the shape of the curve. At all ph values Mn++ is a better Fig. 1. The effect of ph on the activity of NAD : Isocitrate dehzldroaenase.. standard assav conditions: x. plus 5. mm LMP;, with 3. mm MgS4;, the i@3d4replaced by 1 mm &lnso, The Role of NAD and Inhibition by NADH The reaction velocity varies with NAD concentration according to normal MichaelisMenten kinetics except at low isocitrate concentrations. From this it can be deduced that only one molecule of NAD binds to the enzyme as is the case with the Neurospora enzyme [3]. When the isocitrate concentration is held constant at 1 mm, variations in the levels of Mg++ and Mn++ produce small changes in the values of the K, for NAD but when either cation is saturating a K, of about.2mm is observed. The maximum velocity in the presence of Mg++ is 62 Oi of that in the presence of Mn++. The potent competitive inhibition by NADH has already been indicated in earlier reports [1,2]. From plots of the reciprocal of the reaction rate against the NADH concentration at varying levels of NAD the inhibitor constant (Iii) was determined. There is a large difference in the inhibitor constant depending on which cation is the cofactor. In the presence of 1 mri Mg++ the Kf is.5 mm and with 1 mm Mn++ it is.16 mm. NADPII is much less inhibitory than NADH. The initial absorbance of reaction mixtures containing high concentrations of NADPH makes accurate kinetic determinations difficult. However, the Kd for

3 Vol.7, No.2, 1969 'I!. P. COULTATE and D. T. DENNIS 155 NADPH is approximately.5 mm under standard assay conditions. 4 O Inhibitory Action of Nucbotides Five nucleoside triphosphates, ADP, AMP, UMP and adenosine were assayed for inhibition under standard assay conditions. All the triphosphates arc inhibitory although ATP is more inhibitory than the others. ADP is less inhibitory and AMP, UMP and adenosine show practically no inhibition. Since the stabilities of the metal chelate compounds of the nucleotides and nucleoside [16] are paralelled by their inhibitory properties it can be assumed that the inhibition is due primarily to metal withdrawal. Inhibition by Inorganic Anions The inhibition of the enzyme by inorganic anions as described in other reports [2,1] was conhmed. This is shown as an increase in the sigmoid nature of the rate response to isocitrate concentration. However, in contrast to the enzyme from peas, SO,= is also inhibitory. The reversibility of the inhibition by increasing isocitrate concentration was demonstrated. The nonlinearity of double reciprocal plots of rate of reaction versus isocitrate concentration make detailed kinetic analysis difficult. However, in the presence of citrate the enzyme shows normal MichaelisMenten kinetics (see below). Under these conditions sulphate shows strictly competitive inhibition with isocitrate and a value of the K1 for SO,= of 63 mm was obtained. Increasing the concentration of Mg++ or Mn++ reverses the inhibition by sulphate although this is probably an indirect effect. As wiu be shown below increasing the concentration of cation lowers the K, for isocitrate and it is the lowering of the K, for isocitrate which reverses the SO4= inhibition. Effects of Organic Anions A number of organic acids were tested for inhibitory and stimulatory effects over a range of isocitrate concentrations between.2 and 2. mm. Pyruvatc, acetate, crketoglutarate, succinate, L malate and Dmalate at 2 mm have no effect. Oxaloacetate could not be tested because malate dehydrogenase was present in the enzyme preparation. Interactions between Isocitrate and Activating Metal Ions Plots of reaction velocity versus isocitrate concentration at varying magnesium and manganese levels are shown in Fig. 2 A and 2 B. Extrapolation of the curved double reciprocal plots gives values for the maximum velocities. In the case of Mg++ as activator a common value of.43 A units/& is obtained whereas with Mn++ as activator the maximum 1' c m N 2 W 1.. f.6 i2 $ 5 a.4 a e'.3 I/ > 2 1 W A e B Fig.2. (A) The effect of isocitrate concentration on the rate of NAD : Isocitrate dehydrogenuse in the presence of three different concentrations of 5 mn;, 1 mm;,.5 mm. (B) The effect of isocitrate cancentration on the rate of NAD: Isocztrate dehydrogenase in the presence of three concentrations of 1 mm;,.1 mm;,.1 mm velocity is a factor of MnSO, concentration. This discrepancy in behaviour is assumed to be caused by the much lower concentrations of Mn++ becoming limiting. As suggested by Fig. 2 A and 2 B there is a tendency towards MichaelisMenten kinetics as the concentration of either Mg++ or Mn++ is raised. This observation was borne out by an application of the Hill equation to these results as has been described by Atkinson and coworkers [7]. Double reciprocal plots of activity versus MgSO, or MnSO, concentration were derived from the data of Fig.2A and 2B. These plots were straight lines

4 156 Regulation of a Plant NAD: Isocitrate Dehydrogenasc European J. Biochem. 2 2 LL A B Fig.3. (A) The effect of ieocitrate concentration on the K, for MnSO,. (B) The effect of isocitrate concentration of the I(, for MgSO, c? B LL w LL 2 c& 2 I I 1 I OO IMgS43(rnM) tmns4l(mm) Fig. 4. The effect of cation concentration on the Km for isocitrate suggesting only one cation binding site and also showed that saturating Mi++ gave a higher maximum velocity than saturating Mg++. The K, values for the cations obtained were then plotted against isocitrate concentration as shown in Fig.3A and 3B. In both cases elevation of the concentration of isocitrate lowers the Km for the cation to a limiting value which is about.1 mm for MgSO, and.7 mm for MnSO,. A similar effect has been noted for the yeast [S] and insect flight muscle [8] enzymes. The isocitrate concentrations which gave half maximum velocities were taken as the apparent Km values. As shown in Fig.4A and 4B limiting values of the K, for isocitrate of about.35mm in the presence of Mg++ and.25mm in the presence of Mn++ are reached. Other Bivalent Cations as Activators Besides Mg++ and Mn++, Zn++ and Co++ are found to activate the enzyme. Ca++ is not an activator of the enzyme and it does not inhibit in the presence of.2 mm MnSO, (a concentration which almost saturates the enzyme). The activities of the five cations are compared in Fig.5. The inhibition by Zn++ at elevated concentrations was examined in the presence of.2mm MnSO, and found to be reversed by citrate and elevated concentrations of isocitrate. The inhibition could not be reversed by increasing the NAD or Mn++ concentrations. It is suggested that zinc ions inhibit the enzyme by removal of the isocitrate as a zinc complex. Activation by Citrate The enhancement of activity by citrate has been noted previously [l, 21. The optimum concentration for citrate stimulation is 1 mm. Above this concentration the stimulatory effect is masked by citrate inhibition. Elevation of either magnesium or isocitrate readily reverses this inhibition. The amount of enhancement depends largely on degree of satura

5 Vo1.7, No.2, 1969 T. P. COULTATE and D. T. DENNIS 157 absence of the 2 or 3bydroxyl group activates the enzyme in a similar manner to citrate. A double reciprocal plot of activation versus activator concentration at.5 mm isocitrate in the presence of 5 mm Na,SO, (Fig.6) indicates Km values of.5 mm and 2.5 mm for citrate and tricarballylate respectively. Sdphate was added to the incubation mixture to increase the stimulatory effect of citrate and tricarballylate. It should be noted, however, that tricarballylate at high concentrations is much less inhibitory than citrate. [CATIONI (mm) Fig.5. The activation of NAD : isocitrate dehydrogenase by various cations 111 ISOCITRATE OR TRICARBALLY LATE 1 WmM) Fig.6. A double reciprocal plot of citrate and tricarballylate activation of NAD : Isocitrate dehydrogenase in the presence of.5 mm isocitrate. The velocity, v, is the velocity in the presence of activator and v is the velocity in the absence of activator tion of the enzyme with isocitrate. When there are only low concentrations of inorganic anions saturation with isocitrate is nearly complete at 1 mm and so no appreciable enhancement by citrate occurs. Citrate like isocitrate reverses the inhibition by inorganic anions. Tricarballylate (propane 1,2,3tricarboxylic acid) which differs from citrate and isocitrate only in the DISCUSSION The data presented here show that the enzyme from swede, while differing to some extent from the enzyme from fungi and animal tissues, does have the properties of a regulatory enzyme. Comparison with the enzyme from other sources shows it generally to have less complex regulatory properties. The potent inhibition by NADH is in accord with the fhdings for the Neurospora enzyme [4,6] and also bears out the observation of Chen and Plaut [lo] that the enzyme has a much greater affinity for NADH than NAD. They found a twofold difference between the values of Kt for NADH and Km for NAD in the presence of 1.33 mm MnCl,. The swede enzyme exhibits a similar difference (the ratio of Kc to Km is.67) in the presence of 1. mm MnSO, but replacement of manganese ions by magnesium ions reduces the Ka for NADH from.16 to.5 mm and raises the K, for NAD from.24 to.44 mm so that the Kz: Km ratio is.114. Since neither AMP nor ADP have any positive effector action on the swede enzyme the importance of the NAD:NADH ratio as a regulatorymechanismis heightened. The NAD :NADH ratio will reflect the ATP: ADP ratio by the operation of the electron transport chain. Since the tricarboxylic acid cycle is producing other metabolites besides ATP the regulation of the cycle by the NAD : NADH ratio may be more logical. The observed inhibition by ATP is probably due to metal chelation. It has been suggested that such chelating effects could be important in regulation [17] but this would be an indirect and less specific means of regulation. High concentrations of inorganic anions are inhibitory and the inhibition is apparently competitive with isocitrate. The relief of sulphate inhibition by increased concentrations of Mg++ and Mn++ is probably a result of enhanced binding of isocitrate. A similar explanation may apply to the observation by Cennamo and coworkers that inhibition of the yeast enzyme by inorganic anions was relieved by AMP [12]. Since inorganic anions are unlikely to occur in the mitochondria in the concentration required for the inhibition described in this paper, they probably have little importance naturally. However, an inhibited form such as this may represent

6 158 T. P. COULTATE and D. T. DENNIS : Regulation of a Plant NAD : Isocitrate Dehydrogenase European J. Biochem. the state of the enzyme in the cell. It is possible that many regulatory enzymes are normally in an inhibited state in the cell. It has been suggested that phosphofructokinase is in an inhibited state in the cell because the ATP would be at an inhibitory concentration [18,19]. The significance ofthe cation cofactor has received little attention in previous reports although from the results presented here the cation cofactor is obviously of great importance. The Km for Mn++ is much lower than the K, for Mg++ and t,he apparent K, for isocitrate is dependent on both the type and concentration of cation. Similarly the Kt for NADH is lower with Mg++ than with Mn++. The maximum velocity is also higher in the presence of Mn++. It has been suggested that isocitrate and metal bind independently and not as the preformed complex [12]. They also appear to bind at the same site [12]. It is probable that the ternary complex of enzyme, cation and isocitrate is more stable than the binary complexes with the enzyme. Increasing the concentration of either cation or isocitrate will facilitate the formation of the ternary complex and hence lower the K, for the other component. It is also possible that the ternary complex with Mn++ is more stable than that with Mg++ which would explain the fact that Mn++ is a better cofactor than Mg++. It has been suggested by Atkinson and coworkers [7] that citrate can bind at both isocitrate sites so that at low concentrations is acts as a positive effector but at high concentrations it is a competitive inhibitor with isocitrate. Since the inhibition at high citrate concentrations can be reversed by increasing themg++ concentration it is highly probable that the inhibition by citrate is due to metal chelation. It may be concluded from Fig.6 that one molecule of citrate or tricarballylate (and by inference one molecule of isocitrate) binds at the allosteric site and in the presence of citrate the enzyme binds one molecule of isocitrate, namely that at the catalytic site. Tricarballylate which also activates shows much less inhibition and is also a less effective metal chelator [16]. Tricarballylate has also been shown to activate fatty acid biosynthesis [2]. The allosteric site appears to require a tricarboxylic acid since dicarboxylic acids such as succinate have no effect. Citrate will be important as a regulator since the equilibrium position ofthe aconitase reaction is in favour of citrate [21]. REFERENCES 1. Dennis, D. T., and Conltate, T. P., Life Sci. 6 (1967) Cox, G. F., and Davis, D. D., Biochem. J. 15 (1967) Sanwal, B. D., Stachow, C. S., and Cook, R. A., Biochemistry, 4 (1965) Sanwal, B. D., and Stachow, C. S., Biochim. Biophys. Actu, 96 (1965) Sanwal, B. D., and Cook, R. A., Biochemistry, 6 (1966) Hathaway, J. A., and Atkinson, D. E.: J. Biol. Chem. 238 (1963) Atkinson, D. E., Hathaway, J. A., and Smith, E. C., J. Biol. Chem. 24 (1964) Klingenberg, &I., Goebell, H., and Wenske, G., Biochem (1965) Chen, R. F., and Plaut, G. W. E., Biochemistvy, 2 (1963) Cennamo, C., Montecuccoli, G., and Bonaretti, G., Biochim. Biophys. Actu, 11 (1965) Cennamo, C., Montecuccoli, G., and Bonaretti, G., Boll. SOC. Ital. Biol. Sper. 42 (1966) Cennamo, C., Montecuccoli, G., and Bonaretti, G., Biochim. Biophys. Actu, 132 (1967) Chen, R. F., Brown, D. M., and Plaut, G. W. E., Biochemistry, 3 (1964) Cennamo, C., Montecuccoli, G., and Bonaretti, G., Boll. SOC. Ital. Biol. Sper. 42 (1966) Lowry,. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J., J. Bio2. Chem. 193 (1951) Sillen, L. G., and Martell, A. E., Stability Constulzts of Hetul Ion Complexes, The Chemical Society, London Wyatt, H. V., J. Theoret. Biol. 6 (1964) Mansour, T. E., J. Biol. Chem. 238 (1963) Dennis, D. T., and Coultate, T. P., Biochim. Biophys. Actu, 146 (1967) Fang, M., and Lowenstein, J. M., Biochem. J. 15 (1967) m England, P. J., Denton, R. M., and Randle, P. J., Biochem. J. 15 (1967) 32C. T. P. Coultate Department of Biochemistry of the University Leicester, Great Britain D. T. Dennis Department of Biology, Queen s University Kingston, Ontario, Canada