Glutamate-malate metabolism in liver mitochondria. A model constructed on the basis of mitochondrial levels of enzymes, specificity, dissociation constants, and stoichiometry of hetero-enzyme complexes.

The level of aspartate aminotransferase in liver mitochondria was found to be approximately 140 microM, or 2-3 orders of magnitude higher than its dissociation constant in complexes with the inner mitochondrial membrane and the high molecular weight enzymes (M(r) = 1.6 x 10(5) to 2.7 x 10(6)) carbamyl-phosphate synthase I, glutamate dehydrogenase, and the alpha-ketoglutarate dehydrogenase complex. The total concentration of aminotransferase-binding sites on these structures in liver mitochondria was more than sufficient to accommodate all of the aminotransferase. Therefore, in liver mitochondria, the aminotransferase could be associated with the inner mitochondrial membrane and/or these high molecular weight enzymes. The aminotransferase in these hetero-enzyme complexes could be supplied with oxalacetate because binding of aminotransferase to the high molecular weight enzymes can enhance binding of malate dehydrogenase, and binding of both malate dehydrogenase and the aminotransferase facilitated binding of fumarase. The level of malate dehydrogenase was found to be so high (140 microM) in liver mitochondria, compared with that of citrate synthase (25 microM) and the pyruvate dehydrogenase complex (0.3 microM), that there would also be a sufficient supply of oxalacetate to citrate synthase-pyruvate dehydrogenase.     

Substrate channeling of NADH and binding of dehydrogenases to complex I

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.     

Binding of malate dehydrogenase and NADH channelling to complex I

As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochodrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the completing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channeling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction.     

The interaction of yeast citrate synthase with yeast mitochondrial inner membranes

The specific interaction of yeast citrate synthase with yeast mitochondrial inner membranes was characterized with respect to saturability of binding, pH optimum, effect of ionic strength, temperature response, and inhibition by oxalacetate. The binding ability of the inner membranes is inhibited by proteolysis and heat treatment, which implies that the membrane component(s) responsible for binding is a protein. A protein fraction from inner membranes when added to liposomes will bind citrate synthase. In addition, the binding of yeast fumarase, mitochondrial malate dehydrogenase, and cytosolic malate dehydrogenase to yeast inner membranes was examined. For these studies the yeast mitochondrial matrix enzymes, citrate synthase (from two types of yeast), malate dehydrogenase, and fumarase, as well as cytosolic malate dehydrogenase, were purified using rapid new techniques.     

Towards the molecular basis for the regulation of mitochondrial dehydrogenases by calcium ions

In mammalian cells, increases in calcium concentration cause increases in oxidative phosphorylation. This effect is mediated by the activation of four mitochondrial dehydrogenases by calcium ions; FAD-glycerol 3-phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol 3-phosphate dehydrogenase, being located on the outer surface of the inner mitochondrial membrane, is exposed to fluctuations in cytoplasmic calcium concentration. The other three enzymes are located within the mitochondrial matrix.While the kinetic properties of all of these enzymes are well characterised, the molecular basis for their regulation by calcium is not. This review uses information derived from calcium binding studies, analysis of conserved calcium binding motifs and comparison of amino acid sequences from calcium sensitive and non-sensitive enzymes to discuss how the recent cloning of several subunits from the four dehydrogenases enhances our understanding of the ways in which these enzymes bind calcium. FAD-glycerol 3-phosphate dehydrogenase binds calcium ions through a domain which is part of the polypeptide chain of the enzyme. In contrast, it is possible that the calcium sensitivity of the other dehydrogenases may involve separate calcium binding subunits.     

Acrolein is a mitochondrial toxin: effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Acrolein is an air pollutant from cigarette smoking and other pollutions and also a by-product of lipid peroxidation. Studies have demonstrated that acrolein causes cytotoxicity and genotoxicity, including liver damage and death of hepatocytes. However, the toxic effects and the underlying mechanisms of acrolein on mitochondria, especially, on liver mitochondria, have not been well studied. In the present study, we investigated the toxic effects and mechanisms of acrolein on mitochondria isolated from rat liver by examining mitochondrial respiration, dehydrogenases, complex I, II, III, IV and V, permeability transition, and protein oxidation. Acrolein incubation (10-1000 microM, or 0.02-2 micromol/mg protein) with mitochondria caused dose-dependent inhibition of NADH- and succinate-linked mitochondrial respiration chain, change of mitochondrial permeability transition, increase in protein carbonyls, and selective enzyme inhibition of mitochondrial complex I, II, pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, but no effects on mitochondrial complex III, IV, V and malate dehydrogenase. These results suggest that acrolein is a mitochondrial toxin and that mitochondrial dysfunction caused by acrolein may play an important role in acrolein toxicity such as hepatotoxicity and also smoking-related diseases.     

Regulation of mitochondrial dehydrogenases by calcium ions

Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.     

Regulation of malate dehydrogenase activity by glutamate, citrate, alpha-ketoglutarate, and multienzyme interaction

Binding experiments indicate that mitochondrial aspartate aminotransferase can associate with the alpha-ketoglutarate dehydrogenase complex and that mitochondrial malate dehydrogenase can associate with this binary complex to form a ternary complex. Formation of this ternary complex enables low levels of the alpha-ketoglutarate dehydrogenase complex, in the presence of the aminotransferase, to reverse inhibition of malate oxidation by glutamate. Thus, glutamate can react with the aminotransferase in this complex without glutamate inhibiting production of oxalacetate by the malate dehydrogenase in the complex. The conversion of glutamate to alpha-ketoglutarate could also be facilitated because in the trienzyme complex, oxalacetate might be directly transferred from malate dehydrogenase to the aminotransferase. In addition, association of malate dehydrogenase with these other two enzymes enhances malate dehydrogenase activity due to a marked decrease in the Km of malate. The potential ability of the aminotransferase to transfer directly alpha-ketoglutarate to the alpha-ketoglutarate dehydrogenase complex in this multienzyme system plus the ability of succinyl-CoA, a product of this transfer, to inhibit citrate synthase could play a role in preventing alpha-ketoglutarate and citrate from accumulating in high levels. This would maintain the catalytic activity of the multienzyme system because alpha-ketoglutarate and citrate allosterically inhibit malate dehydrogenase and dissociate this enzyme from the multienzyme system. In addition, citrate also competitively inhibits fumarase. Consequently, when the levels of alpha-ketoglutarate and citrate are high and the multienzyme system is not required to convert glutamate to alpha-ketoglutarate, it is inactive. However, control by citrate would be expected to be absent in rapidly dividing tumors which characteristically have low mitochondrial levels of citrate.     

Absence of NADH channeling in coupled reaction of mitochondrial malate dehydrogenase and complex I in alamethicin-permeabilized rat liver mitochondria

A simple in situ model of alamethicin-permeabilized isolated rat liver mitochondria was used to investigate the channeling of NADH between mitochondrial malate dehydrogenase (MDH) and NADH:ubiquinone oxidoreductase (complex I). Alamethicin-induced pores in the mitochondrial inner membrane allow effective transport of low molecular mass components such as NAD+/NADH but not soluble proteins. Permeabilized mitochondria demonstrate high rates of respiration in the presence of malate/glutamate and NAD+ due to coupled reaction between MDH and complex I. In the presence of pyruvate and lactate dehydrogenase, an extramitochondrial competitive NADH utilizing system, respiration of permeabilized mitochondria with malate/glutamate and NAD+ was completely abolished. These data are in agreement with the free diffusion of NADH and do not support the suggestion of direct channeling of NADH from MDH to complex I.     

Supramolecular organization of enzymes of the tricarboxylic acid cycle

In virtue of analysis of data on the interaction of tricarboxylic acid cycle enzymes with the mitochondrial inner membrane and data on the enzyme-enzyme interactions, the spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon) is proposed. The alpha-ketoglutarate dehydrogenase complex, adsorbed on the mitochondrial inner membrane along one of its 3-fold symmetry axes, plays the key role in the formation of metabolon. Two association sites of the alpha-ketoglutarate dehydrogenase complex located on opposite sides of the complex participate in the interaction with the membrane. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleosidediphosphate kinase. Succinate dehydrogenase, the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of metabolon on the membrane. The molecular mass of the complex (ignoring succinate dehydrogenase) is of 8.10(6) daltons. The metabolon symmetry corresponds to the D3 point symmetry group. It is supposed, that the tricarboxylic acid cycle enzyme complex interacts with other multienzyme complexes of the matrix and the electron transfer chain.     

The role of malate in regulating the rate of mitochondrial respiration in vitro

Depletion of endogenous malate by preincubation of mitochondria at 30 degrees C in substrate-free media sharply decreases the rate of citrate oxidation and inhibits mitochondrial respiration in the presence of pyruvate and alpha-ketoglutarate. Addition of catalytic amounts of endogenous malate and its production via succinate oxidation promote rapid oxidation of citrate and pyruvate in the mitochondria and abolishes the lag period with alpha-ketoglutarate Malate increases the rate of membrane potential generation after addition of citrate, pyruvate or alpha-ketoglutarate to mitochondrial suspensions. Studies with controlled malate concentrations revealed that the changes in malate concentrations observed in the mitochondria in the presence of gluconeogenesis-inducing hormones may be due to the influence of these hormones on mitochondrial oxidation.     

Inhibition of mitochondrial alpha-ketoglutarate dehydrogenase by 1-methyl-4-phenylpyridinium ion

Effects of 1-methyl-4-phenylpyridinium ion (MPP+) on the activities of NAD+- or NADP+-linked dehydrogenases in the TCA cycle were studied using mitochondria prepared from mouse brains. Activities of NAD+- and NADP+-linked isocitrate dehydrogenases, NADH- and NADPH-linked glutamate dehydrogenases, and malate dehydrogenase were little affected by 2 mM of MPP+. However, alpha-ketoglutarate dehydrogenase activity was significantly inhibited by MPP+. Kinetic analysis revealed a competitive type of inhibition. Inhibition of alpha-ketoglutarate dehydrogenase may be one of the important mechanisms of MPP+-induced inhibition of mitochondrial respiration, and of neuronal degeneration.     

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.     

Supramolecular organization of tricarboxylic acid cycle enzymes

We propose a spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon). The structure is based on an analysis of data on the interaction between tricarboxylic acid cycle enzymes and the mitochondrial inner membrane, as well as on data on enzyme-enzyme interactions. The alpha-ketoglutarate dehydrogenase complex, adsorbed along one of the 3-fold symmetry axes of the mitochondrial inner membrane, plays a key role in formation of the metabolon. In the interaction with the membrane, two association sites of the alpha-ketoglutarate dehydrogenase complex participate, placed on opposite sides of the complex. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleoside-diphosphate kinase. Succinate dehydrogenase, which is the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of the metabolon on the membrane. The molecular mass of the complex (without regard to succinate dehydrogenase) is 8 x 10(6) Da. The metabolon symmetry corresponds to the D3 point symmetry group.     

Binding of adducts of NAD(P) and enolizable ketones to NAD(P)-dependent dehydrogenases

Carbonyl compounds such as alpha-ketoglutarate, pyruvate, oxaloacetate, butyraldehyde, acetaldehyde or acetone react with NAD or NADP to give adducts. Binding studies of adducts to dehydrogenases are performed by means of ultraviolet differential spectroscopy, circular dichroism and spectrofluorimetry. The dehydrogenases show a high degree of binding specificity toward the adducts which contain their specific oxidized substrate and their specific coenzyme. The high selectivity of the dehydrogenases for adducts is evidenced by binding studies of NAD(P)-pyruvate and NAD(P)-alpha-ketoglutarate adducts on glutamate dehydrogenase at pH 7.6 and 8.9. Evidence is presented showing that adducts bind to the active site of the enzymes.     

Organization of Krebs tricarboxylic acid cycle enzymes

Binding of enzymes of the Krebs TCA cycle to biological membranes was characterized with respect to intracellular location, susceptibility to various chemical and physical treatments, and extractability as a macromolecular component of the mitochondrial inner membrane. It was shown that citrate synthase and malate dehydrogenase bind to the inner membrane in an ionic strength-sensitive, saturable, and specific manner to a relatively thermostabile component manifested on the inner (matrix) surface of the inner membrane of the mitochondrion. From these data several arguments in support of the physiological applicability of these processes were deduced, and the question of whether these two enzymes bind to the same or different membrane components was considered. Also, experiments preliminary to purification of the citrate synthase binding component were presented.     

Keto acid metabolism in Desulfovibrio.

Four strains of Desulfovibrio each excreted pyruvate to a constant level during growth; it was re-absorbed when the substrate (lactate) was exhausted. Malate, succinate, fumarate and malonate also accumulated during growth. One of the strains (Hildenborough) excreted alpha-ketoglutarate as well as pyruvate when incubated in nitrogen-free medium; the former was re-absorbed on addition of NH4Cl. In a low-lactate nitrogen-free medium, strain Hildenborough rapidly re-absorbed the pyruvate initially excreted, but did not re-absorb the alpha-ketoglutarate. Arsenite (I mM) prevented the accumulation of alpha-ketoglutarate; I mM-malonate did not affect the accumulation of keto acids. Isocitrate dehydrogenase activity (NAD-specific) in all strains was lower than NADP-specific glutamate dehydrogenase activity. Alpha-Ketoglutarate dehydrogenase could not be detected in any strain. NADPH oxidase activity was demonstrated. This and previous work indicate that a tricarboxylic acid pathway from citrate to alpha-ketoglutarate exists in Desulfovibrio spp., and that succinate can be synthesized via malate and fumarate; however, an intact tricarboxylic acid cycle is evidently not present. The findings are compared with observations on biosynthetic pathways in clostridia, obligate lithotrophs, phototrophs, and methylotrophs, and various facultative bacteria.     

Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.     

Transport of pyruvate and lactate in yeast mitochondria.

Evidence for the existence of mediated transport of pyruvate and lactate in isolated mitochondria of Saccharomyces cerevisiae is presented. 1. The mitochondrial oxidation of pyruvate is specifically inhibited by the monocarboxylic oxoacids alpha-ketoisocaproate and by alpha-cyano-3-hydroxycinnamate, while pyruvate and malate dehydrogenases activities are not inhibited. 2. The stimulation of the mitochondrial oxidations of succinate, alpha-ketoglutarate and citrate by pyruvate are also inhibited by alpha-cyano-3-hydroxycinnamate. 3. The [14C]pyruvate uptake by yeast mitochondria follows saturation kinetics and is completely inhibited by alpha-cyano-3-hydroxycinnamate. 4. Large amplitude passive swellings of mitochondria of the wild type and of cytoplasmic rho- and rho-n mutants are induced by isoosmotic ammonium pyruvate and lactate. These pH-dependent swellings are inhibited by alpha-cyano-3-hydroxycinnamate suggesting that the carrier system is not coded by mitochondrial DNA.     

Citrate Cycle and Related Metabolism of Listeria monocytogenes

The growth response of Listeria monocytogenes strains A4413 and 9037-7 to carbohydrates was determined in a defined medium. Neither pyruvate, acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, nor malate supported growth. Furthermore, inclusion of any of these carbohydrates in the growth medium with glucose did not increase the growth of Listeria over that observed on glucose alone. Resting cell suspensions of strain A4413 oxidized pyruvate but not acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, or malate. Cell-free extracts of strain A4413 contained active citrate synthase, aconitate hydratase, isocitrate dehydrogenase, malate dehydrogenase, fumarate hydratase, fumarate reductase, pyruvate dehydrogenase system, and oxidases for reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. The alpha-ketoglutarate oxidation system, succinate dehydrogenase, isocitrate lyase, and malate synthase were not detected. Cytochromes were not detected. The data suggest that strain A4413, under these conditions, utilizes a split noncyclic citrate pathway which has an oxidative portion (citrate synthase, aconitate hydratase, and isocitrate dehydrogenase) and a reductive portion (malate dehydrogenase, fumarate hydratase, and fumarate reductase). This pathway is probably important in biosynthesis but not for a net gain in energy.