Interaction between Citrate Synthase and Malate Dehydrogenase

Abstract

The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase. The interactions between pig heart citrate synthase and mitochondrial malate dehydrogenase or cytosolic malate dehydrogenase were studied using the frontal analysis method of gel filtration and by precipitation in polyethylene glycol. This method showed that an interaction between citrate synthase and mitochondrial malate dehydrogenase occurred but no interaction between citrate synthase and cytosolic malate dehydrogenase. Channeling of oxaloacetate in the malate dehydrogenase and citrate synthase-coupled systems was tested using polyethylene glycol precipitates of citrate synthase and mitochondrial malate dehydrogenase, and citrate synthase and cytosolic malate dehydrogenase. The effectiveness of large amounts of aspartate aminotransferase and oxaloacetate decarboxylase, as competing enzymes for the intermediate oxaloacetate, was examined. Aspartate aminotransferase and oxaloacetate decarboxylase were less effective competitors for oxaloacetate when precipitated citrate synthase and mitochondrial malate dehydrogenase in polyethylene glycol was used at low ionic strength compared with free enzymes in the absence of polyethylene glycol or with a co-precipitate of citrate synthase and cytosolic malate dehydrogenase. Substrate channeling of oxaloacetate with citrate synthase-mitochondrial malate dehydrogenase precipitate was inefficient at high ionic strength. These effects could be explained through electrostatic interactions of mitochondrial but not cytosolic malate dehydrogenase with citrate synthase. mitochondrial malate dehydrogenase citrate synthase mitochondrial aconitase oxaloacetate polyethylene glycol aspartate aminotransferase yeast mitochondrial CS yeast mitochondrial MDH OAA decarboxylase cytosolic MDH 5,5′-dithiobis(2-nitrobenzoic acid). In the last twenty years it has been demonstrated that sequential enzymes which operate within a metabolic pathway interact with each other to form highly organized complexes (1Srere P.A. Annu. Rev. Biochem. 1987; 56: 89-124Crossref PubMed Scopus (807) Google Scholar, 2Ovádi J. Cell Architecture and Metabolic Channeling. R. G. Landes Co., Austin, TX1995Google Scholar). The term metabolon was introduced to describe such enzyme-enzyme complexes (3Srere P.A. Trends Biochem. Sci. 1985; 10: 109-110Abstract Full Text PDF Scopus (240) Google Scholar, 4Robinson Jr., J.B. Srere P.A. J. Biol. Chem. 1985; 260: 10800-10805Abstract Full Text PDF PubMed Google Scholar). Among the mitochondrial enzymes of the Krebs tricarboxylic acid cycle, interactions occur between six of eight sequential enzymes: fumarase with mitochondrial malate dehydrogenase (mMDH)1 (5Beeckmans S. Driessche V.E. Kanarek L. Eur. J. Biochem. 1989; 183: 449-454Crossref PubMed Scopus (25) Google Scholar), mMDH with citrate synthase (CS) (6Srere P.A. Mattiasson B. Mosbach K. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2534-2538Crossref PubMed Scopus (159) Google Scholar, 7Halper L.A. Srere P.A. Arch. Biochem. Biophys. 1977; 184: 529-534Crossref PubMed Scopus (80) Google Scholar, 8Tompa P. Batke J. Ovádi J. Welch G.R. Srere P.A. J. Biol. Chem. 1987; 262: 6089-6092Abstract Full Text PDF PubMed Google Scholar, 9Merz J.M. Webster T.A. Appleman J.R. Manley E.R., Yu, H.-A. Datta A. Ackerson B.J. Spivey H.O. Arch. Biochem. Biophys. 1987; 258: 132-142Crossref PubMed Scopus (8) Google Scholar, 10Datta A. Merz J.M. Spivey H.O. J. Biol. Chem. 1985; 260: 15008-15012Abstract Full Text PDF PubMed Google Scholar, 11Lindbladh C. Rault M. Hagglund C. Small W.C. Mosbach K. Bülow L. Evans C. Srere P.A. Biochemistry. 1994; 33: 11692-11698Crossref PubMed Scopus (69) Google Scholar), succinyl-CoA synthetase with α-ketoglutarate dehydrogenase complex (12Porpaczy Z. Sumégi B. Alkonyi I. Biochim. Biophys. Acta. 1983; 749: 172-179Crossref PubMed Scopus (37) Google Scholar), mitochondrial aconitase (mACO) with CS (13Srere P.A. Sherry A.D. Malloy C.R. Sumégi B. Agius L. Sherratt H.S.A. CHANNELING IN INTERMEDIARY METABOLISM. Portland Press Ltd., London1997: 201-217Google Scholar), NAD-dependent isocitrate dehydrogenase with mACO (14Tyiska R.L. Williams J.S. Brent L.G. Hudson A.P. Clark B.J. Robinson Jr., J.B. Srere P.A. Welch J.R. Clegg J.S. THE ORGANIZATION OF CELL METABOLISM. 1986: 177-189Crossref Google Scholar), and NAD-dependent isocitrate dehydrogenase with α-ketoglutarate dehydrogenase complex (15Porpaczy Z. Sumégi B. Alkonyi I. J. Biol. Chem. 1987; 252: 9509-9514Abstract Full Text PDF Google Scholar). Moreover, it has been shown that all of these enzymes bind to the inner surface of the mitochondrial inner membrane, whereas purified isozymes from other cellular compartments do not possess such binding abilities (13Srere P.A. Sherry A.D. Malloy C.R. Sumégi B. Agius L. Sherratt H.S.A. CHANNELING IN INTERMEDIARY METABOLISM. Portland Press Ltd., London1997: 201-217Google Scholar). Organized enzyme complexes may have several kinetic advantages for the cell (16Welch G.R. Prog. Biophys. Mol. Biol. 1977; 32: 103-191Crossref PubMed Scopus (175) Google Scholar, 17Spivey H.O. Merz J.M. Bioessays. 1989; 10: 127-130Crossref PubMed Scopus (44) Google Scholar). One possible advantage is preventing intermediates from escaping into solution where they may be sequestered by other enzymes for use in different metabolic pathways. In other words, the close proximity of consecutive enzymes of a metabolic pathway may be used to increase the metabolic flow through this pathway, especially by channeling the substrate at reduced bulk substrate concentration. However, there are methodological problems in demonstrating thein vitro existence of such intermediate channeling within enzyme-enzyme complexes. First, many of these interactions between enzymes are relatively weak, and the isolation of their complexes from cells is difficult because during the isolation procedure dilution effects or changes in the ionic strength will tend to dissociate the complexes. Different strategies have been used to obtain the active sites of consecutive enzymes in close proximity and to assess possible kinetic advantages of such an arrangement. Datta et al. (10Datta A. Merz J.M. Spivey H.O. J. Biol. Chem. 1985; 260: 15008-15012Abstract Full Text PDF PubMed Google Scholar) showed channeling of oxaloacetate (OAA) between mMDH and CS in a polyethylene glycol (PEG) precipitate of the two enzymes by using aspartate aminotransferase (AAT) as a trap for the intermediate OAA. Their results indicate that OAA is channeled between mMDH and CS, because large amounts of AAT are unable to trap OAA in the precipitate of the CS and mMDH. Another approach is the use of genetic fusions of consecutive enzymes, which may indicate the channeling of an intermediate substrate in solution. For instance, recent experimental work of Lindbladh et al. (11Lindbladh C. Rault M. Hagglund C. Small W.C. Mosbach K. Bülow L. Evans C. Srere P.A. Biochemistry. 1994; 33: 11692-11698Crossref PubMed Scopus (69) Google Scholar) on yeast mitochondrial CS (CS1) and yeast mitochondrial MDH (Mdh1p) connected by a short linker sequence into a fusion protein showed the existence of channeling of OAA between these two enzymes using the same competitive assay procedure as Datta et al. (10Datta A. Merz J.M. Spivey H.O. J. Biol. Chem. 1985; 260: 15008-15012Abstract Full Text PDF PubMed Google Scholar). Because of the short length of the linker connecting the CS and MDH enzymes, the orientational possibilities for the enzymes in the fusion protein are reduced, and a molecular model of the complex (11Lindbladh C. Rault M. Hagglund C. Small W.C. Mosbach K. Bülow L. Evans C. Srere P.A. Biochemistry. 1994; 33: 11692-11698Crossref PubMed Scopus (69) Google Scholar) was proposed using the known structure of the porcine enzymes. Elcock and McCammon (18Elcock A.H. McCammon J.A. Biochemistry. 1996; 35: 12652-12658Crossref PubMed Scopus (61) Google Scholar) performed Brownian dynamics simulations to investigate the channeling of OAA in this model and provided evidence for an efficient electrostatically based channeling mechanism of OAA, even though mMDH and CS active sites are separated by nearly 60 Å in the model of the fusion protein. Another support for electrostatic channeling between two enzyme sites is provided by recent experimental work which shows that the efficiency of substrate transfer within dihydrofolate reductase-thymidylate synthase is dependent on the ionic strength (19Trujillo M. Donald R.G.K. Roos D.S. Greene P.J. Santi D.V. Biochemistry. 1996; 35: 6366-6374Crossref PubMed Scopus (62) Google Scholar). The same enzyme modeling strategy was extended with mACO, and recent computer graphic modeling studies provide a putative complex of porcine mACO·CS·mMDH complex (20Vélot C. Mixon M.B. Teige M. Srere P.A. Biochemistry. 1997; 36: 14271-14277Crossref PubMed Scopus (132) Google Scholar). The model shows an electrostatically favorable channel between CS and mACO as well as the already reported mMDH-CS channel. Using genetically prepared fusions with a short linker may not provide correct data about electrostatic channeling because the fusion of CS and mMDH may produce an unphysiological orientation of active sites in these constructs. Elcock et al. (21Elcock A.H. Huber G.A. McCammon J.A. Biochemistry. 1997; 36: 16049-16058Crossref PubMed Scopus (64) Google Scholar) recently developed a kinetic model for a coupled two-enzyme system containing a third enzyme that competes for the common intermediate. They then used the kinetic data of Lindbladh et al. (11Lindbladh C. Rault M. Hagglund C. Small W.C. Mosbach K. Bülow L. Evans C. Srere P.A. Biochemistry. 1994; 33: 11692-11698Crossref PubMed Scopus (69) Google Scholar) for CS1 and Mdh1p and showed for the free coupled enzyme system using free mMDH and CS, in the presence of increasing amounts of the competing enzyme AAT, that the theoretically derived curve matches exactly the experimentally observed data. When the kinetic data for CS1/Mdh1p fusion protein in the presence of AAT are analyzed with their kinetic model, the experimental data fit a theoretical curve that indicates 74% channeling of the OAA in the fusion protein. In this study, we extend the previous work of Datta et al.(10Datta A. Merz J.M. Spivey H.O. J. Biol. Chem. 1985; 260: 15008-15012Abstract Full Text PDF PubMed Google Scholar) to a study of pig heart CS and mMDH in PEG by using the frontal analysis variant of the Hummel-Dreyer (22Hummel J.P. Dreyer W.J. Biochim. Biophys. Acta. 1962; 63: 530-532Crossref PubMed Scopus (931) Google Scholar) method of gel filtration and by studying the kinetics of co-precipitates of these enzymes in PEG solution. Because there is a great difference between conditions generally used to characterize biochemical reactions in vitro and conditions that actually exist in the living cells, the use of crowding agents such as PEG make it possible to induce the formation of enzyme complexes in vitro with conditions which reproduce the crowding effect in cells. To ensure that the use of AAT as an OAA trap does not give special results, we also used a second OAA trapping enzyme, OAA decarboxylase (OAADC). By means of these two different OAA trapping reactions, the substrate channeling of OAA within the PEG-precipitated CS and mMDH was demonstrated. No substrate channeling of OAA within a PEG precipitate of CS and cytosolic MDH (cMDH) was detected using the trapping method. We also report the effect of ionic strength on the PEG precipitation and on the OAA channeling of the enzymes. The following enzymes were used: pig heart mMDH and pig heart cMDH (EC 1.1.1.37), pig heart CS (EC 4.1.3.7), AAT (EC 2.6.1.1), and OAADC (EC 4.1.1.3) were obtained from Sigma. Malate, NADH, NAD, OAA, DTNB, and glutamate were also from Sigma. PEG (M r = 4000) was purchased from Fluka (Switzerland). Superdex 200 was obtained from Pharmacia LKB (Sollentuna, Sweden). All other chemicals used were of the highest purity available. Salt and buffer exchanges from the commercial enzyme suspensions were made by using Sephadex G-10 columns just prior to the experiments. CS activity was determined using 0.2 mm acetyl-CoA, 0.5 mm OAA, and 0.1 mm DTNB in 100 mm Tris-HCl (pH 7.5) (23Srere P.A. Brazil H. Gonen L. Acta Chem. Scand. 1963; 17: S129-S134Crossref Google Scholar). The reaction was followed spectrophotometrically at 412 nm. MDH was assayed using 0.1 mm NADH and 0.1 mm OAA in 50 mm Tris-HCl (pH 7.5) at 340 nm (24Englard S. Siegel L. Methods Enzymol. 1969; 13: 99-106Crossref Scopus (103) Google Scholar). AAT was assayed with 30 mm aspartate, 2.5 mm α-ketoglutarate, 0.05 mm pyridoxal phosphate, 0.4 mm DTNB, 0.1 mm acetyl-CoA, and excess pig CS in 100 mmpotassium phosphate buffer (pH 7.5) at 412 nm (25Itoh H. Srere P.A. Anal. Biochem. 1970; 35: 405-410Crossref PubMed Scopus (9) Google Scholar). OAADC was assayed with 40 mm triethanolamine, 9.5 mm Tris-HCl (pH 8.0), 0.5 mm MnCl2, 0.3 mm NADH, 2.3 mm OAA, 9.2 units/ml lactate dehydrogenase (26Dimroth P. Eur. J. Biochem. 1981; 115: 353-358Crossref PubMed Scopus (59) Google Scholar). Extinction coefficients were assumed to be 13,600 and 6300m−1 cm−1 for DTNB and NADH, respectively. One unit of enzyme liberates 1 μmol of product/min. All reactions were measured at room temperature. The overall reaction of malate to citrate catalyzed by MDH and CS, either free or as a PEG CS-MDH precipitate, was monitored in 10 mm malate, 4 mm NAD, and 0.1 mm acetyl-CoA using 0.4 mm DTNB at 412 nm in 10 mm potassium phosphate buffer (pH 8.1). Either AAT (10 units) or OAADC (10 units) were used as a competing enzyme for OAA. For AAT, 10 mm glutamate was added to the reaction mixture. For OAADC, the assay was performed in a Tris-HCl buffer with 1 mm MnCl2. Reactions were measured at 10 °C. Enzyme concentrations are indicated in the text. In 30% PEG, the rate of the coupled reaction was one-fourth that in the absence of PEG. The rate of CS in 30% PEG was similarly decreased. This is in agreement with the report by Datta et al. (10Datta A. Merz J.M. Spivey H.O. J. Biol. Chem. 1985; 260: 15008-15012Abstract Full Text PDF PubMed Google Scholar). The rates of AAT and OAADC were not affected by 30% PEG. The gel filtration column was a Superdex 200 of 0.7 × 30 cm pre-equilibrated in 10 mm potassium phosphate buffer (pH 8.0) with 10% PEG. The optical density of the eluted solution was monitored at 280 nm with a Pharmacia LKB optical unit UV-1. The enzymes, mMDH and cMDH, were added to the column individually to establish their elution volume. The column was then equilibrated by the same buffer containing CS (0.2 mg/ml). Solutions of mMDH and cMDH were injected into the pre-equilibrated column to determine whether a stable complex was formed. The column was thermostatted at 4 °C, and the flow rate was 0.1 ml/min. Fractions (0.5 ml) were collected, and the activities of CS and MDH were determined as described above. MDH-CS complexes were prepared by incubating the two enzymes at 2 mg/ml in 10 mm potassium phosphate buffer (pH 8.0) containing 10 or 25% of PEG for mMDH and cMDH, at 10 for 1 the was at for at 4 °C, a which was then in 30% PEG. frontal analysis method of gel filtration in PEG was used for enzyme-enzyme interaction between CS and the of The column of Superdex 200 was equilibrated with 10 mmpotassium phosphate buffer (pH 8.0) and 10% PEG. mMDH then was added to the shown in 1 the frontal elution of mMDH was about The column was then equilibrated with the same buffer containing 0.2 mg/ml of When a of 0.2 mg/ml of CS and 0.1 mg/ml of mMDH was to the the frontal elution of mMDH was to 1 which indicated complex formation between CS and mMDH. the conditions used for this no precipitate was in the of CS and mMDH that eluted from these The same was performed with the same of cMDH 1 The presence of CS not the elution of the of the frontal elution of mMDH in the presence of CS indicated a interaction between mMDH and The column was with CS, and in the presence of 10% PEG 1 the elution of the same in 100 mm potassium phosphate buffer PEG. the of the PEG CS-MDH complexes prepared as described was of the precipitated CS-MDH complexes 2 were in a with the different PEG concentrations or and ionic (10 or 200 mm for 2 and the of CS-MDH complexes was followed by the in at nm be that the CS-MDH complexes dissociate when PEG at nm However, when the of the enzymes was in 30% PEG at low ionic no in was the ionic strength at 30% PEG a of the of not the 2 for kinetic about of the precipitate in the at high ionic strength 2 The same were performed with complex 2 These results demonstrated that complexes of and were stable at 30% PEG even at high ionic This made it possible to study the kinetic of these complexes. To determine whether there was a kinetic advantage of the coupled systems enzyme in PEG compared with free enzymes, two different enzymes were used for the coupled CS-MDH enzyme In the coupled the rate of of malate to citrate OAA catalyzed by the enzymes MDH and CS was tested in the presence of either excess AAT or excess The same of enzymes was used for all by activity for or of the coupled enzyme reaction with the OAA is shown in This coupled system was used as a for channeling of the intermediate OAA between the enzymes CS and First, AAT was added to the reaction or in PEG and the ionic strength of the reaction different kinetic of the coupled systems of and reaction PEG no complex the coupled reaction the of was reduced to by AAT in the and reduced to for the which showed trapping of the OAA by AAT at low ionic strength. In 30% PEG in which a precipitate for as well as the coupled reaction was reduced to by AAT in channeling of OAA from mMDH to However, the coupled reaction rate was reduced to by AAT for the precipitate which indicated no channeling of OAA. 30% PEG, an increase in ionic strength to 200 mm in a in the coupled reaction by AAT in the complex compared with that in 10 to the with free enzymes. However, with the ionic strength not the coupled reaction AAT was already trapping the OAA that was not results were obtained when OAADC was used as the enzyme of on the MDH used and the ionic strength of the reaction different effects of the of trapping enzymes were observed on the kinetics of coupled systems with and low ionic strength and in the presence of 30% PEG in the reaction a difference in the coupled reaction was observed on the MDH In the of the coupled reaction was reduced to of the but was reduced to of the in the of increase in the ionic strength to 200 mm in a of channeling in the coupled reaction in the presence of OAADC in the of whereas no effect was observed of an increase in ionic strength with the is a difference between conditions generally used to characterize biochemical reactions in vitro and conditions that actually exist in living biochemical reactions are studied in vitro in buffer However, the of a living system from these For as shown for conditions are with many that are and organized in complexes. conditions in the of and of A.P. Anal. 1997; Scholar, J. J. Biophys. J. Full Text PDF PubMed Scopus Google Scholar, A.P. Annu. Rev. Biophys. PubMed Scopus Google Scholar). are that of is affected by less an of by and the of molecular between the active sites of enzymes sequential reactions A.P. Anal. 1997; Scholar, J. J. Biophys. J. Full Text PDF PubMed Scopus Google Scholar, A.P. Annu. Rev. Biophys. PubMed Scopus Google Scholar). The use of crowding such as PEG, the of J. Biol. Chem. 1981; Full Text PDF PubMed Google Scholar, A. PubMed Scopus Google Scholar, Biochem. J. 1963; PubMed Scopus Google Scholar) which also the formation of enzyme-enzyme conditions in The and extended evidence for the interaction between CS and mMDH in PEG. The frontal analysis method of gel filtration showed that the elution of mMDH was when the column was equilibrated with CS When mMDH was with cMDH in these the elution not the of the interaction between the CS and mMDH in PEG be that the elution of mMDH and cMDH CS in PEG were the of the column The in the presence of PEG also elution PEG. elution of in gel filtration in the presence of PEG in absence may be explained by interactions between PEG and the and the gel Biochem. 1985; PubMed Scopus Google Scholar). The interaction of CS and mMDH to form an enzyme-enzyme complex in PEG was by Srere L.A. Srere P.A. Arch. Biochem. Biophys. 1977; 184: 529-534Crossref PubMed Scopus (80) Google Scholar), showed that the of CS and mMDH in PEG in a precipitation of the two enzymes, in to with other enzymes or to a with CS and The results reported demonstrated that the enzyme-enzyme complexes between CS and mMDH obtained by the precipitation in PEG not dissociate at high PEG even for low concentrations of CS and mMDH. increase in ionic strength the of but the complexes in a for at 2 for the of enzyme kinetics of coupled This a study of the kinetics of these complexes. Datta et al. (10Datta A. Merz J.M. Spivey H.O. J. Biol. Chem. 1985; 260: 15008-15012Abstract Full Text PDF PubMed Google Scholar) demonstrated that the coupled reaction catalyzed by the complex of is less to the AAT trapping of OAA the free enzyme coupled which indicates channeling of this intermediate by the of AAT that the citrate reaction rate with free mMDH and CS by do not rates with amounts of the mMDH-CS The results with the previous In this study showed of the OAA intermediate of the complex using OAADC as an trapping enzyme for OAA. These results that the channeling of OAA in the precipitate of it from the trapping by large of AAT and We also that the precipitate was by AAT and OAADC, in that the trapping was the same as that of free enzymes. The is that the kinetic advantages of complexes were because of channeling of substrate OAA from mMDH to CS and not because of the enzymes in a precipitate and the of OAA. theoretical (18Elcock A.H. McCammon J.A. Biochemistry. 1996; 35: 12652-12658Crossref PubMed Scopus (61) Google Scholar) work that the substrate channeling of OAA is because of the electrostatic of the surface of the between the two active and the efficiency of substrate transfer is by electrostatic The results reported on ionic strength effects provide evidence for the theoretical simulations that substrate channeling in is because of electrostatic The for and for We also for