A new procedure has been developed for the isolation of fumarase (EC 4.2.1.2). It is described for the purification of pig heart and liver enzyme. Pyromellitic acid has been covalently coupled to Sepharose-4B with diaminopropanol as spacer arm. When a dialysed 0.55 saturated ammonium sulphate precipitate is applied to the column, in Tris-acetate buffer, pH 7.3, fumarase remains quantitatively bound. It is eluted by competition, together with a few other proteins, by the natural product L-malate. Malate is removed from the eluate by dialysis. After this highly efficient purification step the enzyme is very easily crystallized. The final yield is 67 % for both pig heart and liver preparations. The specific activity of fumarase purified from both tissues is found to be the same.Polyacrylamide gel electrophoresis in dodecylsulphate shows one single band corresponding with a subunit molecular weight of 48 500. A single band is also obtained by electrophoresis in acid urea. This new procedure based on biospecific affinity chromatography allows a fast and easy preparation of gram quantities of fumarase.The enzyme fumarase catalyses the reversible hydration of fumaric acid to L-malic acid. It is involved in the mitochondria1 citric acid cycle as well as in many metabolic processes which occur in the cytoplasm.An easy purification method has been reported earlier for pig heart fumarase by Kanarek and Hill [l]. The critical step in this preparation is the ability to crystallize this enzyme from a rather impure solution (about 1 %), i.e. immediately after the ammonium sulphate steps (0.35 -0.55 saturation). However, when other organisms are considered this procedure usually fails and even with extracts from pig liver, crystallization does not occur.We now report a simple and fast purification technique based on affinity chromatography, using a column with pyromellitic acid as affinity ligand, coupled to the classic Sepharose-4B matrix. The method is described here for pig heart and liver preparations. Purification of the enzyme from other organisms has been successful and will be reported elsewhere. EXPERIMENTAL PROCEDURE MatevialsPig hearts and livers were immediately chilled in ice or frozen at the slaughterhouse. Sepharose-4B EHZJWX. Fumarase, fumarate hydratase (EC 4.2 1.2) was obtained from Pharmacia Fine Chemicals. Pyromellitic acid was from Merck and was used without further purification. l-Cyclohexyl-3-(2-morpholinoethy1)carbodiimide metho-p-toluenesulfonate was from Aldrich. N,N-Dimethylformamide (Merck) and 2-mercaptoethanol (Fluka AG) were freshly distilled before use. Urea solutions were freed from cyanates by passing them through an Amberlite MB-3 column (Rohm-Haas Comp.) ; final concentrations were calculated by way of refractive index. Acrylamide and N,N '-methylene-bisacrylamide were purchased from Fluka AG ; the latter was recrystallized from chloroform. L-Malate and Trizma base (Tris) were from Sigma Chemical Co. All other reagents were 'pro analysis' grade and used without further purification.All pH me...

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 mitochondria) and cytoplasmic halves of the aspartate‐malate shuttle (aspartate aminotransierase 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 metabolism of the existence of a large citric acid cycle/malate‐aspartate shuttle multienzyme complex are discussed.

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A kinetic and ligand binding study on maize (Zea mays) malate synthase is presented. It is concluded from kinetic measurements that the enzyme proceeds through a ternary-complex mechanism. Michaelis constants (Km,glyoxylate and Km,acetyl-CoA) were determined to be 104 microM and 20 microM respectively. C.d. measurements in the near u.v.-region indicate that a conformational change is induced in the enzyme by its substrate, glyoxylate. From these studies we are able to calculate the affinity for the substrate (Kd,glyoxylate) as 100 microM. A number of inhibitors apparently trigger the same conformational change in the enzyme, i.e. pyruvate, glycollate and fluoroacetate. Another series of inhibitors bearing more bulky groups and/or an extra carboxylic acid also induce a conformational change, which is, however, clearly different from the former one. Limited proteolysis with trypsin results in cleavage of malate synthase into two fragments of respectively 45 and 19 kDa. Even when no more intact malate synthase chains are present, the final enzymic activity still amounts to 30% of the original activity. If trypsinolysis is performed in the presence of acetyl-CoA, the cleavage reaction is appreciably slowed down. The dissociation constant for acetyl-CoA (Kd,acetyl-CoA) was calculated to be 14.8 microM when the glyoxylate subsite is fully occupied by pyruvate and 950 microM (= 50 x Km) when the second subsite is empty. It is concluded that malate synthase follows a compulsory-order mechanism, glyoxylate being the first-binding substrate. Glyoxylate triggers a conformational change in the enzyme and, as a consequence, the correctly shaped binding site for acetyl-CoA is created. Demetallization of malate synthase has no effect on the c.d. spectrum in the near u.v.-region. Moreover, glyoxylate induces the same spectral change in the absence of Mg2+ as in its presence. Nevertheless, malate synthase shows no activity in the absence of the cation. We conclude that Mg2+ is essential for catalysis, rather than for the structure of the enzyme's catalytic site.

In recent years, evidence has been accumulating that metabolic pathways are organized in vivo as multienzyme clusters. Affinity electrophoresis proves to be an attractive in vitro method to further evidence specific associations between purified consecutive enzymes from the glycolytic pathway on the one hand, and from the citric acid cycle on the other hand. Our results support the hypothesis of cluster formation between the glycolytic enzymes aldolase, glyceraldehydephosphate dehydrogenase, and triosephosphate isomerase, and between the cycle enzymes fumarase, malate dehydrogenase, and citrate synthase. A model is presented to explain the possibility of regulation of the citric acid cycle by varying enzyme-enzyme associations between the latter three enzymes, in response to changing local intramitochondrial ATP/ADP ratios.