Fur J Blochein. 117. 527-535 (1981) c FEBS 1981 Demonstration of Physical Interactions between Consecutive Enzymes of the Citric Acid Cycle and of the Aspartate-Malate Shuttle A Study Involving Fumarase, Malate Dehydrogenase, Citrate Synthase and Aspartate Aminotransferase Sonia BEECKMANS and Louis KANAREK Laboratorium voor Chemie der Proteinen, Vrije Universiteit Brussel (Received September 15, 198O/Fehruary 26, 1981) By means of covalently immobilized fumarase and mitochondrial or cytoplasmic malate dehydrogenase we were able to detect physical interactions between different enzymes of the citric acid cycle (fumarase with malate dehydrogenase, malate dehydrogenase with citrate synthase and fumarase with citrate synthase) and between the enzymes of both mitochondrial and cytoplasmic halves of the aspartate-malate shuttle (aspartate amino- transferase and malate dehydrogenase). The interactions between fumarase and malate dehydrogenase were also investigated by immobilizing one enzyme indirectly through antibodies bound to Sepharose - protein A. Our results are consistent with a model in which maximally four molecules of malate dehydrogenase are bound to one fumarase molecule. This complex is able to bind either citrate synthase or aspartate aminotransferase. We propose that these enzymes bind alternatively, in order to allow the cell to perform citric acid cycle or shuttle reactions, according to its needs. The physiological meaning and implications on the regulation of me- tabolism of the existence of a large citric acid cycle/malate-aspartate shuttle multienzyme complex are discussed. Whereas it was generally assumed for many years that the Several publications have appeared lately pointing indeed citric acid cycle enzymes are randomly dispersed in the mito- to the existence of physical interactions between enzymes of chondrial matrix, there are in recent literature various publi- the citric acid cycle and aspartate-malate shuttle [7- 141. cations predicting the organization of these enzymes as a Furthermore, a certain compartmentation of different mito- multienzyme complex [I -41. Such predictions were mainly chondria] matrix enzymes has been observed [15] pointing based on the observation that the concentration of free oxalo- to a loose association of certain of the citric acid cycle enzymes acetate is so low that the calculated rate of the citrate syn- with the inner membrane. thase reaction is much slower than the experimentally deter- In this publication we present further indications of real mined citric acid cycle rate as measured by mitochondrial physical interactions between the consecutive citric acid cycle oxygen consumption. Moreover, oxaloacetate further appears enzymes, fumarase, malate dehydrogenase, citrate synthase to be a key metabolite in other important metabolic routes and between the shuttle enzymes malate dehydrogenase and in mitochondria : besides its direct participation in the citric aspartate aminotransferase of both compartments. Fumarase acid cycle and aspartate-malate shuttle it also strongly regu- was included in our experiments as it could be the anchor: lates succinate dehydrogenase activity, whereas it is mainly it links the next enzymes of the citric acid cycle to succinate synthesized by pyruvate carboxylase. It is also important in dehydrogenase, the only enzyme which is located within the cytoplasm: besides operating in the other half of the aspar- mitochondrial inner membrane [I 61 and which connects the tate-malate shuttle, it is the starting product of gluconeo- citric acid cycle with the respiratory chain. Malate dehydro- genesis as the substrate of phosphoenolpyruvate carboxy- genase catalyzes the only citric acid cycle reaction with a kinase [5,6]. Thus channeling on oxaloacetate metabolism highly unfavorable equilibrium malat late]/ [oxaloacetate] and physical association of previously called ‘soluble en- 2 lo4 [17,18]; this forces the cell to make sure that the end zymes’ might be suggested in both cellular compartments. product, oxaloacetate, is removed extremely rapidly in order The advantage of the organization of the citric acid cycle to secure proper functioning of the cycle. Moreover, citrate enzymes as a complex would be the creation of a special synthase, the enzyme next to malate dehydrogenase, is con- microcnvironment around the cycle : the cell would acquire sidered to be the main control point of the cycle [19,20]. One the possibility to maintain a high flux of substrate through could imagine that, besides extensive control by different the cycle with a moderate number of intermediate molecules. substances on this enzyme itself, the fact of switching on and __ ~~ off a physical interaction between malate dehydrogenase and Enzymes. Fumarase or fumarate hydratase (EC 4.2.1.2); malate citrate synthase would be an extra way of regulating the dehydrogenase (EC 1.1.1.37); aspartate aminotransferase (EC 2.6.1.1); cycle activity, especially with the equilibrium of the former citrate synthase (EC 4.1.3.7); aldolase or fructose-bisphosphate aldolase (EC 4.1.2.13); lysozyme (EC 3.2.2.17); succinate dehydrogenase (EC reaction lying far to the left. Moreover, many metabolites 1.3.99.1); pyruvate carhoxylase (EC 6.4.1.1); phosphoenolpyruvate car- which regulate the activity of citrate synthase in vitro (ATP, boxykinase (EC 4.1.1.32); argininosuccinatc lyase (EC 4.3.2.1); fumaryl- pyridine nucleotides and tricarboxylate compounds) have acetoacetase (EC 3.7.1.2); adenylosuccinate iyase (EC 4.3.2.2); glutamate been proven to be much less or even not at all effective dehydrogenase (EC 1.4.1.3). in vivo [21-241. Another question is how the cell manages 528 to separate the amount of oxaloacetate provided to flux enzyme which causes a rate of change of absorbance of through the cycle, from the oxaloacetate which has to operate 0.1 min-l at 25 "C under the experimental conditions. Specific in the shuttle. One possibility is the existence of two pools activities are 1300 units/mg for both mitochondrial aspartate of mitochondrial malate dehydrogenase, and consequently at aminotransferase from chicken and the cytoplasmic isoenzyme least two pools of oxaloacetate in the mitochondria. This from pig hearts; A:%",,,, = 14 1351. supposition is less probable, since the relative amounts of Citrate synthase activity was determined by titration of aspartate aminotransferase and cycle enzymes are constant the released sulphydryl groups of coenzyme A with 5,5'-di- in the mitochondria of all tissues in an organism [25,26]. thiobis(2-nitrobenzoate) as described by Moriyama and Srere Another possibility is regulation by promoting or inhibiting, [37]. One unit of citrate synthase is defined as the amount according to the needs of the cell, a direct interaction of of enzyme that catalyzes the formation of 1 pmol coenzyme A/ aspartate aminotransferase with the malate dehydrogenase min. The specific activity was 160 units/mg; A;% nm, cm = 16. involved in the cycle, thus forming a large combined citric acid cycle/aspartate-malate shuttle complex. Immobilization of Enzymes on Sepharose 48 Four different immobilized enzyme systems were designed MATERIALS AND METHODS with respectively pig fumarase, chicken fumarase, pig mito- chondrial malate dehydrogenase and pig cytoplasmic malate Purifications of Enzymes dehydrogenase covalently coupled to Sepharose 4B. The en- Fumarase from pig hearts [27] and from chicken hearts zymes to be coupled (20 mg of each) were dialyzed first against [28] was prepared as described previously. Mitochondria1 0.01 M potassium phosphate pH 8.2 and afterwards against malate dehydrogenase and mitochondrial aspartate amino- 0.1 M sodium bicarbonate pH 8.2 containing 0.02 M L-malic transferase from chicken hearts were prepared by affinity acid in order to protect the enzymes during the coupling step. chromatography on Sepharose-pyromellitic acid and blue The enzyme solutions were finally adjusted to a concentration Sepharose as will be described elsewhere. Mitochondrial of 1 mg/ml. For each enzyme, 10 g (wet weight) of Sepharose malate dehydrogenase from pig hearts was obtained from 4B (which corresponds to about 10 ml of packed gel), exten- Sigma Chem. Comp. and freed of minor impurities by per- sively washed with distilled water, was suspended in 20ml forming the same affinity chromatographic steps as used for distilled water and activated with 2 g of cyanogen bromide, the chicken heart preparation. Cytoplasmic malate dehydro- suspended in a minimal volume of freshly distilled dimethyl- genase and cytoplasmic aspartate aniinotransferase from pig formamide, during 8 min according to the classical method hearts were from Sigma Chem. Comp. Citrate synthase from of Cuatrecasas [38]. The activated Sepharose was washed chicken hearts was purified as described elsewhere [28]. The quickly with 1 I of cold 0.1 M NaHC03 pH 8.2 and resus- enzyme from pig hearts was obtained from Sigma Chem. pended in the 20 ml enzyme solution. Coupling of the enzymes Comp. All these enzymes were stored at 4°C as ammonium was achieved by overnight shaking of the suspension at 4 "C. sulphate precipitate in 0.01 M potassium phosphate buffer The gels were well washed with 1 1 of 0.1 M NaHC03 and pH 7.3. stored at 4 'C in the presence of sodium azide (200 mg/l All enzymes were tested for purity by electrophoresis in buffer) in order to prevent bacterial growth. the presence of sodium dodecylsulphate [29], by electro- phoresis at pH 8.3 according to Davis [30], by isoelectric Immunological Techniques focusing in Ampholine gradients pH 3.5 - 10 and by electro- phoresis in 6 M urea at pH 3.2 [31] and in 6 M urea at Antibodies against pig heart fumarase and against pig pH 8.0 [32]. Moreover, in neither of these enzyme prepara- heart mitochondrial malate dehydrogenase were raised in tions could we detect any contaminating activity of other rabbits as follows. A solution of 2 mg/ml enzyme was prepared citric acid cycle or aspartate-malate shuttle enzymes. in 0.01 M potassium phosphate buffer pH 8.0 and an emulsion Lysozyme and aldolase were obtained from Boehringer. was made with an equal volume of complete Freund's adju- vant.