A is transferred from acetoacetyl-CoA to succinate, 1 malonic semialdehyde, 1° and malonate, although malonate reacts 50 times slower than succinate, s Succinyl-S-pantetheine, suceinyl-S-glutathione, and acetoacetyl-S-pantetheine are inactive as substrates for the enzyme. 1 Effect o] pH. With succinyl-CoA and acetoacetate as substrates the enzymatic reaction rate increases with p H from pH 7.0 to pH 9.1.1 With acetoacetyl-CoA and suecinate as substrates the reaction shows little dependence on pH between pH 8.1 and pH 8.7. 6 Kinetics and Mechanism of Action. Reciprocal plots of rate against substrate concentration at varying concentrations of the second substrate exhibit parallel lines for the reaction in both directions, i.e., the reaction follows "ping-pong ''11 kinetics? Product inhibition patterns and quantitative studies of the acetoacetate-acetoacetyl-CoA and succinate-succinyl-CoA exchange reactions catalyzed by this enzyme suggest the twostep mechanism shown in Eqs. (1) and (2). E d- acetoacetyl-CoA ~- E • • acetoacetyl-CoA ~ E-CoA d- acetoacetate
E-CoA d- succinate ~ E • • succinyl-CoA ~ E ~ succinyl-CoA
The separation of the overall reaction into its two component half reactions and the isolation of an enzymatically active enzyme-coenzyme A intermediate provide additional support for this mechanism. 6 l~W. W. Cleland, Biochim. Biophys. Acta 67, 104, 173, 188 (1963).
 Succinate Dehydrogenase [EC 126.96.36.199
Succinate: (acceptor) oxidoreductase]
By C. VE~-~ER, D. V. DERVARTANIAN, and W. P. ZEYLEMAKER
Assay M e t h o d In previous volumes of this series methods of assay were described for the particle-bound I and the soluble enzyme, 2 as well as a method of purification of the enzyme from beef heart mitochondria 2 and M~rococcus l~ctilyticus3 Enzyme prepared according to several methods described in the literature 2-4 cannot be used to reconstitute the suceinate oxidase activity of a Keilin and Hartree 5 heart muscle preparation where I W. D. Bonner, Vol. I . 2p. Bernath and T. P. Singer, ¥oi. ¥, p. 82. s T. P. Singer, E. B. Kearney, and P. Bernath, J. Biol. Chem. o.9-3,599 (1956). ~T. Y. Wang, C. L. Tsou, and Y. L. Wang, Sci. Sinica Peking 5, 73 (1956). *D. Keilin and E. F. Hartree, Proc. Roy. Soc. London B129, 277 (1940).
REACTIONS ON THE CYCLE
this activity had been destroyed by alkali treatment2 ,~ A preparation which is capable of reconstituting succinate oxidase is described below. *,8 It is similar to the preparation of Keilin and King 6 and to a modification of the method of Wang and co-workers.4 As outlined in the literature, 8,9 there is no relationship between activities measured with the artificial electron acceptors 1.-0 and the more labile reconstruction activity. Therefore, it has been suggested that reconstitution activity be used as an indicator of the native state of the soluble enzyme. Two methods are generally used for the determination of the activity of soluble succinate dehydrogenase with artificial elcctron acceptors. The first method, a manometric one with phenazine methyl sulfate (PMS), can ' ~. modified for spectrophotometric use although the use of a light-sensitive acceptor requires certain precautions. It has been claimed, s that the rates obtained with the spectrophotometric modification are not proportional with the enzyme concentration. However when a suitable control lacking succinate is used the rates are closely proportional. Nevertheless in our experience the second method, which uses K3Fe(CN)e as acceptor, is more convenient as a routine assay. The method of determination of the reconstitution activity of a soluble, purified succinate dehydrogenase preparation is also described. M a n o m e t r i c M e t h o d with Phenazine M e t h y l Sul]ate :,'° Reagents
Phosphate buffer, 0.3 M, pH 7.6 Succinate, 0.4M, pH 7.6 Bovine serum albumin in H20, 3% (w/v) Cyanide, 30 mM neutralized Phenazine methyl sulfate (PMS) in H20, 1 ~ (w/v), carefully protected from light Enzyme, in oxygen-free 30 mM phosphate buffer containing 0.1% bovine serum albumin, diluted to give an uptake between 2 and 7 t~l of 02 per minute in the assay Procedure. Add to tile main compartments of five Warburg vessels phosphate buffer, 0.5 ml; succinate, 0.3 ml; bovine serum albumin, 0.! ml; enzyme and H=0 to a final volume of 3 ml. Different amounts of PMS are pipetted to the side arms of the vessels; recommended amounts
D. Keitin and T. E. King, Proc. Roy. Soc. Lo~don B152, 163 (1960). 7T. E. King, J. Biol. Chem. 236, 2342 (1961). ST. E. King, J. Biol. C]~ern. 238, 4032 (1963). oT. E. King, J. Biol. Chem. 238, 4037 (1963). ~°E. B. Kearney and T. P. Singer, J. Biol. Chem. 219, 963 (1956).
are 0.2, 0.1, 0.07, 0.05, and 0.04 ml (concentration range, 2.2-0.43 raM). Cyanide, 0.1 ml, is added last. Each vessel is connected immediately to its manometer with the stopcock closed, then placed in the water bath at 38 ° (the pressure is released by opening the stopcock). After 7 minutes' equilibration, the contents are tipped and the oxygen uptake is recorded in the interval 2-7 minutes after tipping. The activity is calculated from double reciprocal plots of activity against dye concentration. In this determination one mole of succinate reduces one mole of oxygen. In the spectrophotometric adaptation of this method, all reagents are used in the amounts described, as well as 0.1 ml of 0.15 mM 2,6-dichlorophenol-indophenol (~ ~-- 21 )< 103 M -l'sec -1 at 600 mt~). Add the reagents to a cuvette thermostatted at 38 ° and start the reaction by adding an amount of enzyme that gives a change in extinction of 0.02-0.1 per minute measured as initial rate. A blank rate (all reagents except succinate) must be determined separately. In this determination 1 mole of succinate reduces 1 mole of dye. Spectrophotometric Method with KsFe(CN)~ (footnotes 8, 11) This method is a modification of the method of Slater and Bonner. 1,12 Reagents Phosphate buffer, 0.3 M, pH 7.6 EDTA, 30 mM pH 7.6 KCN, 0.03 M, neutralized Succinate, 0.4 M, pH 7.6 Bovine serum albumin in H20, 3% (w/v) K3Fe(CN)6, 75 mM stored in a dark bottle Enzyme in oxygen-free 30 mM phosphate buffer containing 0.1% bovine serum albumin, diluted to give a change in extinction of 0.02-0.08 per minute, measured as initial rate Procedure. Add to a spectrophotometer cuvette thermostatted at 25°: H20 to a final volume of 2.9 ml; phosphate buffer, 1 ml; EDTA, 0.1 ml; succinate, 0.3 ml; bovine serum albumin, 0.1 ml; and K3Fe(CN)6, 0.2 ml; 0.1 ml of KCN is added only when particle-bound enzyme is assayed. After noting the extinction at 455 mt~ (c-----150M-~'cm-1), start the reaction by addition of the enzyme, and follow the change in extinction during the first 2 minutes. Initial rates are taken as a measure of activity. A blank rate (all reagents except succinate) must be determined separately. 11D. V. DerVartanian and C. Veeger, BiocMm. Biophys. Act(t 92, 233 (1964). i~E. C. Slater and W. D. Bonner, Biochem. J. 52, 185 (1952).
REACTIONS ON THE CYCLE
In this determination 1 mole of succinate reduces 2 moles of KaFe(CN)6. Concentrations of K~Fe (CN)e of 0.2-5 mM can be used for the calculation of maximal velocities from double reciprocal plots. Concentrations of K~Fe(CN)6 above 5 mM are inhibitory. At low concentrations of K3Fe(CN)~ rates can be measured by following the reaction at 420 m~ (c ~ 1.03 X l0 s M -l"cm-1).
Succinate Oxidase o] Heart Muscle Particles. Reconstitution of the oxygen uptake of particles deprived of succinate oxidase activity. Reagents Phosphate buffer, 0.3 M, pH 7.6 Succinate pH 7.6, 0.4 M EDTA, 30 mM, neutralized Cytochrome c 1% (w/v), in H20 Heart muscle preparation prepared as described below under A of this section Cytochrome c-deficient heart muscle preparation prepared as described under purification procedure Alkali-treated heart muscle preparation, prepared as described under B of this section Soluble succinate dehydrogenase purified up to the gel eluate stage of the purification procedure. A. PREPARATION OF THE HEART MUSCLE PREPARATION OF KEILIN AND
HARTREE2'7'13 The procedure is similar to the one described for preparation of starting material for the soluble enzyme, except that the meat mince is washed only with tap water. B . PREPARATION OF THE
ALKALI-TREATED HEART MUSCLE PREPARA-
The pH of a heart muscle preparation as described under A, protein concentration approximately 10 mg/ml, is adjusted to pH 9.3 with 1 N NaOH. The mixture is incubated for 90 minutes at a temperature of 38 °. After 90 minutes the mixture is cooled to room temperaturc and adjusted to pH 7.6 by careful addition of 1 N HCI. This preparation can be stored for 3 days at 0 ° without significant loss in reconstitution activity. Procedure. a. SUCCINATE OXIDASE ACTIVITY. The succinate oxidase activity is measured manometrically in Warburg flasks at 38 ° in a system containing: H20 to a final volume of 3 ml; phosphate buffer, 1 ml; cytochrome c, 0.1 ml; EDTA, 0.1 ml. The enzyme preparation (in main compartment), is either heart particles prepared as described ~ E . C. Slater, Biochem. J. 45, 1 (1949).
under A or B, or the cytochrome c-deficient heart muscle preparation which is the starting material for the soluble enzyme; 0.3 ml of succinate is added to the side arm. After temperature equilibration the reaction is started by tipping the contents from the side arm and the oxygen uptake is recorded. Keilin and Hartree heart muscle preparation has a specific activity of 0.7-1.4 micromoles of succinate oxidized per minute per milligram of protein. The alkali-treated heart muscle particles have a residual succinate oxidase activity averaging below 5% of the original activity. b. RECONSTITUTION OF SUCCINATE OXmASE ACTIVITY. The mixture of H~O, phosphate buffer, eytochrome c, and EDTA in the amounts given under procedure a is added to the main compartment of the Warburg flasks, followed by the alkali-treated heart muscle preparation in a concentration of about 1 mg/ml final volume, and 0.5-1 mg of soluble succinate dehydrogenase; 0.3 ml succinate is added to the side arm. After 7 minutes' temperature equilibrium at 38 °, the reaction is started by tipping the contents from the side arm and the oxygen-uptake is recorded. Maximal restoration of suceinate oxidase activity is obtained with varying ratios of soluble enzyme to heart muscle preparation depending on pretreatment, purity and age of the preparation. Fully reconstituted alkali-treated heart muscle preparation oxidizes 0.2-0.3 mieromole of succinate per minute per milligram of heart muscle protein. Purification Procedure The procedure comprises 2 parts: (A) preparation of cytochrome c-deficient heart muscle preparation; (B) preparation of the soluble enzyme. A few essential precautions have to be taken in part 2 of the procedure to make sure that reproducible preparations are obtained in terms of activity with artificial hydrogen acceptors and reconstitution activity as well as kinetic and spectral properties. Glass-distilled H~O should be used throughout the entire purification procedure, and all steps should be carried out at 0-4 ° in the presence of 1 mM EDTA. It is very important to eliminate as much oxygen as possible by flushing the solutions with pyrogallol-purified N2. It is preferable to perform all purification steps and centrifugations in closed tubes under N2. The whole procedure from the butanol extraction through the second (NH4)2SO~ precipitation takes less than 31/~ hours. Precipitation and fractionation with (NH4)~S0, significantly increase the purity of the enzyme. On the other hand, purification occurs at the expense of the reconstitution activity, the loss of which is not reproducible.
REACTIONS ON THE CYCLE
In studies with the enzyme purified up to the gel eluate stage of the procedure, it must be kept in mind that the enzyme may contain traces of succinate.
A. Preparation of Cytochrome c-Deficient Heart Muscle Preparation Twenty pig hearts are cleaned of fat and connective tissue and lninced in a meat grinder. The mince is washed with 8-10 changes of 30 liters of tap water, for 15 minutes each time, efficient mechanical stirring being used, and then squeezed by hand through cheesecloth. The water of the last change should be light yellow, not pink. The next step is necessary to remove cytochrome c and other soluble hemoproteins which are difficult to remove during the purification of the soluble enzyme. This step is not included in the normal procedure for the Keilin and Hartree heart muscle preparation. The mince is extracted overnight at 0 ° with 20 liters of 0.15 M phosphate buffer pH 7.6 containing 1 mM EDTA, squeezed through cheesecloth, and washed twice with tap water. Grind 800 g of wet mince, 400 ml of 20 mM phosphate pH 7.6 containing 1 mM EDTA, and 600 g acid-washed and neutralized sand at 0 ° in a mechanical mortar (manufacturers: Pascall Engineering Company, Ltd., Crawley, Sussex, England) until a homogeneous paste is obtained, usually for 20 minutes. ~ Add 600 ml of the same cold phosphate buffer and stir for another 5 minutes. The suspension is centrifuged for 15 minutes at 1000 g. Carefully decant the supernatant and after pH adjustment to 5.7 by the addition of 1 N acetic acid, stir for 5 minutes and centrifuge for 15 minutes at 1500 g. Discard the supernatant, wash the precipitate with cold water, and then centrifuge for an additional 15 minutes at 1500 g. Homogenize the precipitate in 1-1.5 liters of 50 mM borate-50 mM phosphate buffer, pH 8.0, final protein concentration 10-15 mg/ml. In the case of a normal Keilin and Hartree heart muscle preparation, the precipitate is homogenized in 0.1 M phosphate buffer pH 7.6. These preparations can be stored for weeks at 0 °. The succinate oxidase activity declines gradually, while the activity with artificial hydrogen acceptors remains almost unaffected.
B. Preparation o] the Soluble Enzyme All steps are carried out in the absence of 02. Step 1. Solubilization and Gel Eluate. The heart muscle preparation is made anaerobic by flushing it with purified N~. Add sodium succinate to a concentration of 40 mM (the color changes to green) and allow the preparation to stand 2-24 hours in a closed bottle at 0% Add to this
preparation one-fifth of its volume of n-butanol (of --20°), and stir o," shake the mixture for 30 minutes while N_o is bubbling through the solution (at 0°). Centrifuge the mixture in closed transparent polyethylene tubes under N., for 20 minutes at 1500 g. After centrifugation three layers are visible: a sediment, a clear yellow-orange middle layer, and a very turbid upper layer. The middle layer is carefully withdrawn by means of an adjustable suction system. Volume is 800-1100 ml. Contamination with the two other layers must be avoided as it leads to impure preparations. Adjust the pH of the extract to 6.0 by the addition of 1 N acetic acid. Add calcium phosphate gel (prepared according to Keilin and Hal~treeTM) to a final concentration of 4 mg/ml. Stir the suspension for 5 minutes, then centrifuge it for 4 minutes at 1000 g. Discard the supernatant, wash the gel once by stirring with deoxygenated water, and centrifuge the suspension at 1000 g for 5 minutes. Discard the supernatant and add 150-200 ml of 80 mM phosphate buffer pH 7.6 to the tubes. The tubes are made anaerobic, closed, shaken for 10 minutes, then centrifuged at 30,000 q for 5 minutes to remove any butanol-denatured protein bound to the gel. The dark brown gel eluate is collected and either used for further purification or stored in sealed polyethylene tubes under liquid N_o, as described in the next section. Step 2. Fractio77ation with (NH,)~_SO,. Adjust the pH of the geleluate to 7.2 with 1N acetic acid and add solid (NH,)~SO, to 65% saturation (450 g per liter), in 5 minutes, while a stream of N2 is flushed over the solution. Centrifuge the mixture for 10 minutes at 23,000 q. Dissolve the precipitate in 30 ml of 0.1 M phosphate buffer pH 7.6. Add 12 ml of a saturated (at 20 °) solution of (NH,)oSO4 adjusted to pH 7.2 with concentrated NH,OH (0.3 saturation). The pH of the (NH4)~SO, solution is measured in a 1 : 10 dilution. This addition takes ,5 minutes and is performed under a stream of N... Centrifuge the mixture for 5 minutes at 23,000 g and discard the precipitate. Add approximately 20 ml of the saturated (NH,)_oS04 solution to the supernatant (0.5 saturation) in the course of 5 minutes under a stream of N2. Centrifuge the mixture at 30,000 g for 5 minutes, discard the supernatant, and remove the last traces of (NH~)2SO, with filter paper under a stream of N.,. Wash the sediment with anaerobic 0.1M phosphate bufer pH 7.6 by carefully placing a few drops of the buffer on top of the pellet and then removing them. Dissolve the precipitate in about 3 ml of 0.1 M phosphate buffer pH 7.6, amt place the solution in a polyethylene tube covered with a self-sealing rubber stopper through which "D. Keilin and E. F. tIartree, Proc. Roy. Soc. London B19.,4, 397 (1938).
REACTIONS ON THE CYCLE
a needle is inserted. The contents of the tube are subjected to 5-6 cycles of evacuation and refilling with N~, ending with the latter. Withdraw the needle and store the solution in liquid N2. Such frozen preparations are stable for at least 3 weeks. Before use, thaw the contents of a tube at 0 ° and then centrifuge under N2 for 5 minutes at 30,000 g to remove a slight turbidity. The various steps are summarized in the table. Properties The amount of flavin has been determined to be 1 mole per 200,000250,000 g of protein at the second (NH,)2SO, fractionation step. In comparison with the other preparations described 2.4 it is estimated that the enzyme at the second (NH~)2S04 step is more than 70% pure. The flavin (FAD) is covalently linked to the protein. 15,16 There are 8 atoms of nonheme iron per mole of flavin,",~s which is twice as much as in other preparations2.4 The enzyme contains 4-8 atoms of labile sulfide per mole of flavin.1''18 The failure of the enzyme containing four nonheme iron atoms to reconstitute oxygen uptake might be due to degradation of the form containing 8 atoms. It also cannot be excluded that the enzyme as isolated here is a mixture of a primary succinate dehydrogenase containing four iron atoms and a labile nonheme iron protein essential for the connection with the respiratory chain. The enzyme is very unstable at room temperature even when kept under N2. Under all conditions the reconstitution activity declines faster than the activity with PMS and K~Fe(CN)6. The reeonstitution activity is fairly stable upon storage under liquid N~; the activity with hydrogen acceptors is also stable under these conditions. When stored at room temperature the absorbance of the enzyme declines slowly over the entire wavelength range. The process is slower under anaerobic conditions, but there is no relation between decline in absorbance and inactivation." About 22% activation is observed upon incubation at room temperature." This is small in comparison with values obtained with another preparation, 3,~9 indicating that this preparation is fully activated. The spectral changes observed with competitive inhibitors ~9-~ which were first ~T. Y. Wang, C. L. Tsou, and Y. L. Wang, Sc/. 8inica Peking 7, 65 (1958). ~*E. B. Kearney, J. Biol. Chem. 235, 865 (1960). " T . E. King, Biochem. Biophys. Res. Commun. 16, 511 (1964). = W. P. Zeylemaker, D. V. DerVartanian, and C. Veeger, Biochim. Biophys. Acta 09, 183 (10~5). ~E. B. Kearney, J. Biol. Chem. ~20, 363 (1957). 2°D. V. DerVartanian and C. Veeger, Biochim. Biophys. Acta 105, 424 (1965). riD. V. DerVartanlan, W. P. Zeylemaker, and C. Veeger, Syrup. Flavins Flavoproteins 8, 183 (1906).
Z er~ 0
e, " ~
REACTIONS ON Tim C'~'CLE
attributed to activation, were found to be due to the formation of two classes of enzyme-inhibitor complexesJ 1 Reduced glutathione and BAL cause marked changes in the absorption spectrum of the enzyme. 2° Besides succinatc, the enzyme also oxidizes L-ch]orosuccinate, L-methyl succinate, D-malatc, and L-malatc. '-'~ D-Chlorosuccinate, D-methyl succinate, malonate, methylene succinate, malcate, acetoacetare, and oxaloacetate are competitive inhibitors. The kinetically estimated K~ values agree well with disassociation constants of spectrally detectable enzyme-inhibitor complexes. ~,~° The catalytic center activity of the enzyme at 25 ° extrapolated to infinite succinate and K3Fe(CN)6 concentrations is 3900 min -~. The same value is obtained at 25 ° at infinite succinate, and PMS concentrations in the spectrophotometric PMSpromoted reduction of 2,6-dichlorophenol indophenol. -~2The kinetic results indicate t h a t the dissociation of fumarate from the reoxidizcd enzyme is the rate-limiting step in the overall reaction. 2~ The catalytic center activity of the reconstituted particles with a limiting amount of soluble enzyme, at 37 ° and with a succinate concentration of 40 mM, has been found to be 10,000 min -~ (footnote 9). In contrast to data reported in the literature, 25-~7 the enzyme exchanges, upon reduction with succinate in D20, its protons with different rates, ~8 which leads to the formation of ( - - ) - ( R ) - s u c c i n a t e acid-d1, meso-succinate-d~., ( - - ) - ( R , R ) - s u c c i n a t c - d 2 , (--)-(R)-succinate-d3 and succinate-d4. Furthermore the formation of ( ~ ) - ( S ) - d e u t e r a t e d succinates could be observed -~s in case succinate-d4 was exchanged in H20. Application for Analytical Purposes As described elsewhere, the purified enzyme is suitable for the microdete~mination of succinate (see this volume ).
52W. P. Zcylemaker and C. Veeger, unpublished results. ~A. Guiditta and T. P. Singer, J. Biol. Chem. 234, 666 (1959). T. E. King, R. L. Howard, D. F. Wilson, and J. C. R. Li, J. Biol. Chem. 237, 2941 (1962). u O. Gawron, A. J. Glaid, and J. Francisco, Biochem. Biophys. Res. Commun. 9, 237 (1964). Hj. Kahn and D. Rittenberg, Biochem. Biophys. Res. Commun. 27, 484 (1967). sTM. Hiifner, L. M. Buckle)', and T. C. Hollocher, Bioc]~em. Biophys. Res. Com.mun. 28, 791 (1967). ssj. R~tey, J. Seibl, D. Arigoni, J. W. Cornforth, G. Ryback, W. P. Zeylemaker, and C. Veegcr, Nature 216, 5122 (1967).