Association between the α-ketoglutarate dehydrogenase complex and succinate thiokinase

Association between the α-ketoglutarate dehydrogenase complex and succinate thiokinase

172 Biochimica etBiophysicaActa, 749 (1983) 172-179 Elsevier BBA31778 ASSOCIATION BETWEEN T H E a-KETOGLUTARATE D E H Y D R O G E N A S E C O M P L...

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172

Biochimica etBiophysicaActa, 749 (1983) 172-179

Elsevier BBA31778

ASSOCIATION BETWEEN T H E a-KETOGLUTARATE D E H Y D R O G E N A S E C O M P L E X AND SUCCINATE T H I O K I N A S E ZOLTAN PORPACZY, BALAZSSOMEGI and ISTVAN ALKONYI Institute of Biochemistry, University Medical School, H-7624 Pecs, Szigeti ut 12 (Hungary)

(ReceivedJune 23rd, 1983)

Key words: a-Ketoglutarate dehydrogenase complex," Succinate thiokinase; Enzyme- enzyme interaction," Citric acid cycle," (Pig heart)

The kinetic parameters of the individual reaction of pig heart a-ketoglutarate dehydrogenase complex, succinate thiokinase and the a-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The Km C°A of a-ketoglutarate dehydrogenase complex and the KSm uccinylC°A of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the a-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the a-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed association with a-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and a-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large muitienzyme complex in the mitochondrial matrix.

Introduction Previously, it was generally believed that the enzymes of the citric acid cycle are randomly dispersed in the mitochondrial matrix. Recently, various reports [1-3] have predicted the organization of citric acid cycle enzymes as a complex. The organization of the citric acid cycle enzymes may be advantageous, allowing a high flux of the citric acid cycle maintained by a moderate number of intermediate molecules. Until now, most of the attention has been focused on the interaction of citrate synthase, or malate dehydrogenase, with some metabolically related enzymes [4-11]. In this paper, we give evidence for the association between a-ketoglutarate dehydrogenase comAbbreviation: DTNB, 5,5'-dithiobis(2-nitrobenzoicacid). 0167-4838/83/$03.00 © 1983 ElsevierSciencePublishers B.V.

plex and succinate thiokinase. As a consequence of the association, the a-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system had higher activity than was expected from the kinetic parameters of the individual enzymes. Since both enzymes are at branching points of the citric acid cycle, the observed interaction raises new possibilities in the regulation of the metabolic pathways involved.

Materials and Methods Enzymes, reagents a n d chemicals. Pig heart citrate synthase (citrate oxaloacetate-lyase ( p r o 3S-CH2COO----, acetyl-CoA), EC 4.1.3.7), pig heart cytosolic malate d e h y d r o g e n a s e (Lmalate : N A D + oxidoreductase, EC 1.1.1.37), Clostridium kluyveri phosphotransacetylase (acetyl-

173

CoA : orthophosphate acetyltransferase, EC 2.3.1.8), pig heart isocitrate dehydrogenase (threoDs-isocitrate : N A D P ÷ oxidoreductase (decarboxylating), EC 1.1.1.42), acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1), coenzyme A, fluorescein isothiocyanate, rhodamine B isothiocyanate, thiamine pyrophosphate, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and albumin (bovine) were obtained from Sigma. Sepharose CL-4B and Sephadex G-25 were from Pharmacia. Pig heart succinate thiokinase (succinate:CoA ligase (GDP-forming), EC 6.2.1.4), lactate dehydrogenase (t-lactate:NAD + oxidoreductase, EC 1.1.1.27) and rabbit muscle pyruvate kinase (ATP : pyruvate 2-O-phosphotransferase, EC 2.7.1.40), were from Boehringer Mannheim GmbH. All other chemicals were of the highest purity commercially available. Enzyme preparation. Highly purified a-ketoglutarate 0ehydrogenase complex (2-oxoglutarate : lipoamide oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2) and pyruvate dehydrogenase complex (pyruvate:lipoate oxidoreductase, (decarboxylating and acceptoracetylating), EC 1.2.4.1) were prepared from pig heart as described by Stanley and Perham [12], and were stored at a concentration of 50-80 units/ml in small batches at -20°C. The specific activities of a-ketoglutarate dehydrogenase complex and pyruvate dehydrogenase complex were 8-11 units/mg protein. The purity of the enzymes was checked with SDS gel electrophoresis. Enzyme assays. The citrate synthase was assayed as described in Ref. 8. The malate dehydrogenase, isocitrate dehydrogenase, phosphotransacetylase and acetate kinase activities were determined as described in Ref. 13. The activity of pyruvate dehydrogenase complex was measured as described [14]. The activity of a-ketoglutarate dehydrogenase complex was determined in the same reaction mixture as the pyruvate dehydrogenase complex except that the pyruvate was replaced by 2 mM a-ketoglutarate. The succinate thiokinase activity was determined as described [151. Kinetic experiments. The overall reaction rate of a-ketoglutarate dehydrogenase complex was determined spectrophotometrically in a 20-mm

0 I

HOOC- C - C~-C~-COOH

NADH ° H+ CO~

NAD ÷

CoA- S,H

GTP ÷ SUCCINATE

5UCCINYL-CoA

OOO + ( ~

Fig. 1. Scheme of the et-ketoglutarate dehydrogenase complexsuccinate thiokinase coupled system. The et-ketoglutarate dehydrogenase complex and succinate thiokinase are denoted by KGDC and STK, respectively.

path-length cuvette, by monitoring the NADH formation at 340 nm. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.8)/2 mM a-ketoglutarate/2 mM MgC12/0.2 mM thiamine pyrophosphate/1 mM NAD+/0.1 mM GDP in a final volume of 1 ml. The concentrations of CoA are specified in Fig. 1. The Km c°A of ct-ketoglutarate dehydrogenase complex was determined from the steady-state rates in both the individual reaction of a-ketoglutarate dehydrogenase complex and the a-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system (Fig. 1). In this latter case, the activities of a-ketoglutarate dehydrogenase complex and succinate thiokinase were 12 m U / m l and 0.8 U/ml, respectively. The KSm"CCinyl'C°A of succinate thiokinase in the individual reaction was determined as described Cha and Parks [16]. In the coupled system, the reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0)/0.02 mM thiamine pyrophosphate/0.1 mM N A D + / 2 mM MgC12/0.2 mM a-ketoglutarate/0.1 mM GDP in a final volume of 1 ml. The concentration of succinyl-CoA is specified in Fig. 2. In this coupled system, the steady-state reaction rate was reached after the first 10-20 s. The CoA formed from succinyl-CoA in the succinate thiokinase reaction was resynthesized to succinyl-CoA by ct-ketoglutarate dehydrogenase complex. Concomitantly, the a-ketoglutarate dehydrogenase complex produced a stoichiometric amount of CO2

174

and NADH. Therefore, the steady-state rate of the coupled system could be determined from the N A D H formation. Since the activities of a-ketoglutarate dehydrogenase complex and succinate thiokinase were 1 U / m l and 0.015 U / m l , respectively, the activity of succinate thiokinase determined the rate of the coupled system. Under this condition, the greatest part of CoA was in the succinylated form (the steady-state C o A / succinyl-CoA ratio was lower than 0.1 as determined from the deproteinized reaction mixture), and therefore this coupled system is applicable for the determination of the g Sm uccinyl'C°A of succinate thiokinase. The reaction rates were corrected by the rate of the chemical hydrolysis of succinyl-CoA taking place under the reaction conditions. The rate of succinyl-CoA hydrolysis was determined in a reaction mixture containing the components of the succinate thiokinase-a-ketoglutarate dehydrogenase complex coupled system except succinate thiokinase. Labelling of proteins with fluorescent dyes. 2 mg of proteins were dissolved in 1 ml 50 mM phosphate buffer, pH 8.0, and were reacted with 40/~g fluorescent dyes/fluorescein isothiocyanate or rhodamine B isothiocyanate for 15 min at 20°C. The reaction was stopped with 100 ~1 of 0.5 M Tris-HC1 buffer, pH 8.0, and the free dye was removed with gel chromatography on a Sephadex G-25 column. The amount of covalently incorporated dye was 1.3-1.6 molecules dye per polypeptide chain. Poly(ethylene glycol)precipitation. Enzymes were dissolved in 0.9 ml 50 mM phosphate buffer, pH 7.0, containing 1 mM MgC12, and 0.1 ml 50% poly(ethylene glycol) was added to this solute. After incubation for 20 min at 22°C, samples were centrifuged at 4°C for 20 rain at 25 000 × g. The supernatant solution was removed and the pellet was dissolved in 1 ml 50 mM potassium phosphate buffer, pH 7.0. The enzyme activities (or fluorescence of labelled proteins) of the original sample, supernatant solutions and resuspended pellets were determined. Enzyme co-sedimentation. The investigated enzyme pairs previously equilibrated against 50 mM potassium phosphate buffer, p H 7.0, containing 1 mM MgC12, were placed into a polyethylene tube and were incubated for 30 rain at 4°C. After

preincubation, tubes were centrifuged for 140 min at 150000 x g. The enzyme activities or fluorescence of labelled proteins were determined in the original enzyme solution, in resuspended pellet and in supernatant. Preparations of a-ketoacid dehydrogenase complex contain a small amount of NADH-oxidase and GTPase activities. Since these enzyme activities interfere with the succinate thiokinase assay, the NADH-oxidase and GTPase activities were determined in the control experiment. These enzyme activities were determined in the succinate thiokinase reaction mixture from which succinate was omitted. Gel chromatography. Migration of succinate thiokinase alone or in the presence of a-ketoglutarate dehydrogenase complex was studied on a Sepharose CL-4B column (1 × 18 cm). 0.1 ml of succinate thiokinase was applied to the top of the column, which had been equilibrated with 50 mM potassium phosphat buffer, pH 7.8, containing 1 mM MgC1 z. The succinate thiokinase was eluted with this buffer alone or with added a-ketoglutarate dehydrogenase complex. The column was thermostatted at 22°C. The flow rate was 12 m l / h and 0.25-ml fractions were collected. Enzyme activities or the fluorescence of labelled proteins were determined in the fractions. Results

Kinetic experiments The dependence of the initial rate of a-ketoglutarate dehydrogenase complex on coenzyme A concentration was studied in the 5 - 1 0 0 / z M concentration range (Fig. 2). The plot of 1/v vs. 1 / C o A was linear in the studied concentration range and the Kmc°A value of a-ketoglutarate dehydrogenase complex was found to be 5.5/zM. This Kmc°A value was near to those reported by others

[17,18]. In the a-ketoglutarate dehydrogenase complexsuccinate thiokinase coupled system (activity of succinate thiokinase >> activity of a-ketoglutarate dehydrogenase complex) almost all CoA was in non-acylated form. Therefore, such a system is appropriate for determining the Kmc°A value of a-ketoglutarate dehydrogenase complex. It can be calculated from the data of Fig. 2 that the K c°A of a-ketoglutarate dehydrogenase complex in the

175

x.., ,'r

0,2

0,2O

/

~ 0,I0 // ! 10o

|

t CoA/*ram"t Fig. 2. Lineweaver-Burk plots with respect to CoA for the a-ketoglutarate dehydrogenasecomplex(e) and a-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system ( × ). The reaction mixture contained 50 mM phosphate buffer (pH 7.8)/2 mM a-ketoglutarate/2 mM MgCI2/0.2 mM thiamine pyrophosphate/1 mM NAD+/0.1 mM GDP and CoA as indicated in the figure in a total volume of 1 ml. The reaction was started with 12 mU a-ketoglutarate dehydrogenase complex. In the case of the a-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system the reaction mixture contained 0.8 U succinate thiokinase. ,~O

coupled system was lower than the K~ °~" of a-ketoglutarate dehydrogenase complex in the individual reaction. The other kinetic parameters of a-ketoglutarate dehydrogenase complex did not change in the coupled system (data not shown). Changing the ratio of enzymes in the coupled system so that activity of a-ketoglutarate dehydrogenase complex >> activity of succinate thiokinase, the dependence of the steady-state reaction rate on the succinyl-CoA c o n c e n t r a t i o n gives the KSmuccinyl-C°A value of succinate thiokinase. In this coupled system, the K SmUCCinyl'C°Avalue of succinate thiokinase was found to be 1.5 /~M (Fig. 3). The K Sm uccinyl'C°A of succinate thiokinase was also determined in the individual reaction and its value was 65 /~M (Fig. 3). That is, the KSm uccinyl'C°A of succinte thiokinase is 30-times less in the coupled system than in the individual enzyme reaction. Moreover, a small increase in the Vmax of succinate thiokinase in the coupled system was also observed when the reaction rate was determined in the succinyl-CoA + G D P + phosphate --. succinate + GTP + CoA direction. These kinetic data suggest interactions between a-ketoglutarate dehydrogenase complex and suc-

I

I

I

L

t ~ - c,A/f~¢~ Fig. 3. Lineweaver-Burkplots with respect to succinyl-CoAfor the individual succinate thiokinase reaction (x) and the a-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system (e). The reaction mixture contained 50 mM phosphate buffer (pH 7.0)/0.2 mM a-ketoglutarate/2 mM MgC12/ 20/~M TPP/0.2 mM NAD+/0.1 mM GDP and succinyl-CoA as indicated in the figure. The activity of a-ketoglutarate dehydrogenase complex was 1 U/ml in the coupled system, while that of succinate thiokinase was 15 mU/ml in both cases. The reaction was started with succinyl-CoA.Details of assay conditions are described under Materials and Methods.

cinate thiokinase that change some of the kinetic parameters of the studied enzymes.

Direct evidence for the interaction Enzyme-enzyme interaction can be detected by co-precipitation of the respective proteins in poly(ethylene glycol) solution [4,6]. Proteins in the absence or in the presence of a-ketoglutarate dehydrogenase complex were precipitated with poly(ethylene glycol) (5% w/v). As can be seen in Table I, significant co-precipitation was observed only in the presence of succinate thiokinase and a-ketoglutarate dehydrogenase, indicating the specific interaction between the two enzymes. Because of the very great molecular weight difference between a-ketoglutarate dehydrogenase complex and succinate thiokinase, the possible association could be studied by ultracentrifugation. Proteins were preincubated alone or in combination with a-ketoglutarate dehydrogenase complex, and centrifuged. As seen in Table II, the a-ketoglutarate dehydrogenase complex carried down succinate thiokinase with itself, while the sedimentation of the other five proteins was negligible in the presence of a-ketoglutarate dehydrogenase complex.

176 TABLE I

TABLE II

INTERACTION BETWEEN ENZYMES IN POLY(ETHYLENE GLYCOL)

CO-SEDIMENTATION OF PROTEINS UNDER THE INFLUENCE OF ULTRACENTRIFUGATION

In these experiments, enzymes in the indicated amounts were incubated for 20 min alone or in the presence of a-ketoglutarate dehydrogenase complex in 1 ml 5% poly(ethylene glycol), and then centrifuged, and the supernatant and precipitate were assayed as described under Materials and Methods. The values represent the mean ± S.E. of three experiments.

Enzymes alone or in the presence of a-ketoglutarate dehydrogenase complex were incubated for 30 min at 4°C and centrifuged for 120 min at 150000×g. After centrifugation, the supernatant and the resuspended pellet were assayed for activities as described under Materials and Methods. The values represent the mean ± S.E. of three experiments.

Enzyme

Enzyme

Percentage of the total activity in the pelle alone

a-Ketoglutarate dehydrogenase complex (1.6 # M) Succinate thiokinase (5.6 #M) Cytosolic malate dehydrogenase (5.7 #M) Citrate synthase (4 #M) Isocitrate dehydrogenase (NADP +-dep) (4.6/~ M) Phosphotransacetylase (4.8/~M)

98 + 2.0

Percentage of total activity in the pellet

in the presence of 1.6/tM a-ketoglutarate dehydrogenase complex a-Ketoglutarate dehydrogenase complex (1.6 # M) Succinate thiokinase (5.6 #M) Cytosolic malate dehydrogenase (5.6 #M) Citrate synthase (4.1 #M) Isocitrate dehydrogenase (NADP +-dep) (6/~ M) Phosphotransacetylase (5.2/~M) Acetate kinase (4.2/~M)

--

0

26.7_+2.0

0

4.7 ± 1.0

0

4.1 ± 1.0

0

2.3 + 0.8

0

4.3 + 1.5

alone

with the a-ketogiutarate dehydrogenase complex (1.6/~M)

98 ± 2.0

--

0

25.4+ 2.0

0

3.2 ± 1.0

0

5.6 ± 1.5

0

2.1 ± 1.0

0

4.2 ± 1.3

0

4.1 ± 1.2

TABLE III PRECIPITATION OF RHODAMINE B-LABELLED AND FLUORESCEIN-LABELLED ENZYME IN THE PRESENCE OF POLY(ETHYLENE GLYCOL) Labelled proteins were precipitated under the same conditions as described in Table I. The amounts of proteins in the supernatant and precipitate were determined fluorimetrically; see Materials and Methods. The values represent the mean ± S.E. of three experiments, and are given as the amount of precipitated protein as a percentage of the total. Labelled proteins

Succinate thiokinase (5 #M) Albumin (5/~M) Is±citrate dehydrogenase (NADP+-dep) (4.6/~M)

Labelling with rhodamine,B

Labelling with fluorescein

alone

with a-ketoglutarate dehydrogenase complex (1 ~M)

alone

with a-ketoglutarate. dehydrogenase complex (1 #M)

1 ± 1.0

58 ± 3.0

1.2±1

57±4.0

2+1.0

11±2.5

2 ±1.3

7±2.0

2 ± 1.1

10 :h 2.0

2 ±1.4

14±2.3

177 TABLE IV CO-SEDIMENTATION OF RHODAMINE B- AND FLUORESCEIN-LABELLED PROTEINS UNDER THE INFLUENCE OF U LTRACENTRIFUGATION Labelled proteins were ultracentrifuged under the same conditions as in Table II. Proteins in the supernatant and in the resuspended pellet were assayed fluorimetrically; see Materials and Methods. The values represent the mean + S.E. of three experiments, and are given as the amount of protein sedimented as a percentage of the total. Labelled proteins

Succinante thiokinase (5 #M) Albumin (5 #M) Isocitrate dehydrogenase (NADP+-dep) (4.6 #M)

Labelling with rhodamine B

Labelling with fluorescein

alone

with a-ketoglutarate dehydrogenase complex (1 #M)

alone

with a-ketoglutarate dehydrogenase complex (1 #M)

0

53+3.0

0

51 +3.0

0

9 + 2.0

0

10 + 1.5

0

10 ___2.0

0

8 + 1.0

In the above experiments, the amounts of sedimented or precipitated enzymes were determined from the enzyme activities (in the supernatant and in the pellet). Labelling of proteins with fluorescence dyes, the interaction between a-ketoglutarate dehydrogenase complex and enzymes or

TABLE V CO-SEDIMENTATION OF SUCCINATE THIOKINASE IN THE PRESENCE OF VARIOUS PROTEINS UNDER THE INFLUENCE OF ULTRACENTRIFUGATION Succinate thiokinase alone and in the presence of various proteins was incubated for 30 min at 4°C and ultracentrifuged for 120 rain at 150000×g. The supernatant and the resuspended pellet were assayed for enzymes as described under Materials and Methods. The values represent the mean+S.E. of three experiments. Enzymes

Succinate thiokinase (% of total) in the pellet in the supernatant

Succinate thiokinase (5.6 #M) Succinate thiokinase (5.6/zM) + a-ketoglutarate dehydrogenase (1.6 #M) Succinate thiokinase (5.6 # M) + pyruvate dehydrogenase complex (0.6/~M) Succinate thiokinase (5.6 #M) + glutamate dehydrogenase (1.74/tM)

0

100

24 +2.5

76 +2.5

8.2+2.2

91.8+2.2

4.9+2.2

95.1+2.2

non-enzyme proteins can be detected by measuring the fluorescence. Association between a-ketoglutarate dehydrogenase complex and labelled proteins was studied (Table III, IV) and a specific interaction between a-ketoglutarate dehydrogenase complex and succinate thiokinase was observed. However, it appears from the data of Tables III and IV that the labelling of proteins with dyes increased both the specific and aspecific interactions. Enzyme-enzyme interactions can be detected with several gel chromatographic techniques [19,20]. In our experiments succinate thiokinase, which was applied to the top of the column, was eluted either with the standard buffer or with the standard buffer containing a-ketoglutarate dehydrogenase complex. The elution profiles of succinate thiokinase (either intact or fluorescein isothiocyanate-labelled) show that the a-ketoglutarate dehydrogenase complex carries forward a portion of succinate thiokinase (Fig. 4). That is, the a-ketoglutarate dehydrogenase complex causes an apparent increase in the molecular weight of succinate thiokinase. It was demonstrated that of the six investigated low molecular weight proteins only succinate thiokinase associated with a-ketoglutarate dehydrogenase complex. However, it would also be of interest to see whether succinate thiokinase associates with other high molecular weight proteins (M r (2-10). 106). Table V shows a

178 v

v

!

I

r

!

"i /0

20

30 F r ~

e

i

40

50

i

,

/lwm~r

i

I00

~0

I0

20

30

40

50

Fig. 4. Influence of a-ketoglutarate dehydrogenase complex o n the elution profile of succinate thiokinase on a column of Sepharose CL-4B. The column (1×18 cm) was equilibrated with 50 mM phosphate buffer, pH 7.4, containing 1 mM MgCl 2. (a) The elution profile of succinate thiokinase was determined by measuring the enzyme activity in the fractions of eluate. 100 ~l succinate thiokinase were developed with either the standard buffer (activity of succinate thiokinase, A) or the standard buffer containing a-ketoglutarate dehydrogenase (activity of succinate thiokinase (e), and a-ketoglutarate dehydrogenase complex (×)). (b) The elution profile of fluorescein isothiocyanate-labelled succinate thiokinase was determined by measuring the fluorescence of the fractions; excitation occurred at 505 nm, emission at 550 nm. 100 /~l of labelled succinate thiokinase were developed with either the standard buffer (fluorescence of succinate thiokinase, A) or with the standard buffer containing a-ketoglutarate dehydrogenase complex (fluorescence of succinate thiokinase (o), and activity of a-ketoglutarate dehydrogenase complex ( × )).

marked precipitation of succinate thiokinase in the presence of a-ketoglutarate dehydrogenase complex, whereas only a slight precipitation was found in the presence of glutamate dehydrogenase and pyruvate dehydrogenase complex. Discussion

In this paper, kinetic and physicochemical evidence is given for the association between a-keto-

glutarate dehydrogenase complex and succiante thiokinase. The association between the two enzymes is proved by gel chromatography, ultracentrifugation and poly(ethylene glycol) precipitation. These experiments show that six other proteins do not associate with c~-ketoglutarate dehydrogenase complex. Moreover, under the conditions employed herein, interaction between succinate thiokinase and c~-ketoglutarate dehydrogenase complex was observed, while significant interactions between pyruvate dehydrogenase complex and glutamate dehydrogenase were not supported. That is, the association between c~-ketoglutarate dehydrogenase complex and succinate thiokinase is mutually specific. Our kinetic data show that the values of Km C°A and K Sm~CCi"Yl'C°gof the respective enzymes are lower in the coupled system than in the individual reactions. That is, in the coupled system, the two enzymes have higher activity than is expected in the knowledge of the kinetic parameters of the individual enzymes. Stere and co-workers [1-3] have postulated previously that the enzymes of the citric acid cycle exist within the matrix of the mitochondrion as a multienzyme complex. The advantage of such an arrangement is obvious in terms of being able to maintain a high flux of substrates through the cycle with a relatively small number of intermediate molecules. Since citrate synthase and malate dehydrogenase are at an important regulatory point of the citric acid cycle and since the mitochondrial oxaloacetate concentration is very low, attention has been mainly focussed on the enzyme-enzyme interactions connected with citrate synthase or malate dehydrogenase. Specific association was detected between citrate synthase and m-malate dehydrogenase in poly(ethylene glycol) solution [6,10]. Moreover, citrate synthase-mmalate dehydrogenase and citrate synthase-mmalate dehydrogenase-fumarase complexes were detected by the means of covalently immobilized fumarase and malate dehydrogenase [11]. However, there are at least two other important regulatory points in the citric acid cycle, isocitrate dehydrogenase and the a-ketoglutarate dehydrogenase complex [21]. Therefore, processes that influence the activity of these enzymes also play a role in the regulation of the flux through the cycle. The association between ~x-ketoglutarate dehydro-

179 g e n a s e c o m p l e x a n d s u c c i n a t e t h i o k i n a s e surely has a n effect o n the free s u c c i n y l - C o A level w i t h i n the m i t o c h o n d r i o n . Since s u c c i n y l - C o A is a n inh i b i t o r of a - k e t o g l u t a r a t e d e h y d r o g e n a s e c o m p l e x [17,221 a n d citrate s y n t h a s e [22,23], the a s s o c i a t i o n b e t w e e n the a - k e t o g l u t a r a t e d e h y d r o g e n a s e c o m plex a n d s u c c i n a t e t h i o k i n a s e p r e s u m a b l y inf l u e n c e s the r e g u l a t i o n of the citric acid cycle at two d i f f e r e n t p o i n t s : at the citrate s y n t h a s e a n d the a - k e t o g l u t a r a t e d e h y d r o g e n a s e c o m p l e x steps. M o r e o v e r , these data, o u r p r e v i o u s o b s e r v a t i o n s [8,9] a n d the results of F a h i e n ' s g r o u p [4,5] i n d i cate that the high m o l e c u l a r weight m i t o c h o n d r i a l c o m p l e x e s are the s t a r t i n g p o i n t s for the f u r t h e r o r g a n i z a t i o n of m i t o c h o n d r i a l e n z y m e s .

References 1 Srere, P.A. (1972) in Energy Metabolism and the Regulation of Metabolic Processes in Mitochondria (Mehlman, M.A. and Hanson, R.W., eds.), pp. 79-91, Academic Press, New York 2 Srere, P.A. and Mosbach, K. (1974) Annu. Rev. Microbiol. 28, 61-83 3 Srere, P.A. (1980) Trends Biochem. Sci. 5, 120-121 4 Fahien, L.A. and Kmiotek, E. (1979) J. Biol. Chem. 254, 5983-5990 5 Fahien, L.A., Kmiotek, E. and Smith, L. (1979) Arch. Biochem. Biophys. 192, 33-46 6 Halper, L.A. and Srere, P.A. (1977) Arch. Biochem. Biophys. 184, 529-534 7 Backman, L. and Johanson, G. (1976) FEBS Lett. 65, 39-43

8 S0megi, B., Gyocsi, L. and Alkonyi, I. (1980) Biochim. Biophys. Acta 616, 158-166 9 Siimegi, B. and Alkonyi, I. (1983) Biochim. Biophys. Acta 749, 163-171 10 Srere, P.A., Halper, L.A. and Finkelstein, M.B. (1978) in Microenvironments and Metabolic Compartmentation (Srere, P.A. and Estabrook, R.W., eds.), pp. 419-432, Academic Press, New York 11 Beekmans, S. and Kanarek, L. (1981) Eur. J. Biochem. 117, 527-535 12 Stanley, C.J. and Perham, R.N. (1980) Biochem. J. 191, 147-154 13 Bergmeyer, H.U. (ed.) (1974) Methods of Enzymatic Analysis, 2nd Edn., Academic Press, New York 14 Siimegi, B. and Alkonyi, I. (1983) Arch. Biochem. Biophys. 223, 417-424 15 Cha, S. (1969) Methods Enzymol. 13, 62-70 16 Cha, S. and Parks, R.E. (1964) J. Biol. Chem. 239, 1968-1877 17 Hamada, M., Koike, K., Nakaula, Y., Hiraoka, T., Koike, M. and Hashimoto, T. (1975) J. Biochem. (Tokyo) 77, 1047-1056 18 McMinn, C.L. and Ottaway, J.H. (1977) Biochem. J. 161, 569-581 19 Winzor, D.J. and Scheraga, H.A. (1963) Biochemistry 2, 1263-1267 20 Jones, M.M., Ogilvie, J.W. and Ackers, G.K. (1976) Biophys. Chem. 5, 339-350 21 Newsholm, E.A. and Staet, C. (1973) Regulation of Metabolism, John Wiley and Sons, London 22 Williamson, J.R., Smith, C.M., LaNoue, K.F. and Bryla, J. (1972) in Energy Metabolic Processes in Mitochondrial (Mehlman, M.A. and Hanson, R.W., eds.), pp. 185-210, Academic Press, New York 23 Smith, C.M. and Williamson, J.R. (1971) FEBS Lett. 18, 35-39