The alpha-ketoglutarate dehydrogenase complex in neurodegeneration

Altered energy metabolism is characteristic of many neurodegenerative disorders. Reductions in the key mitochondrial enzyme complex, the alpha-ketoglutarate dehydrogenase complex (KGDHC), occur in a number of neurodegenerative disorders including Alzheimer's Disease (AD). The reductions in KGDHC activity may be responsible for the decreases in brain metabolism, which occur in these disorders. KGDHC can be inactivated by several mechanisms, including the actions of free radicals (Reactive Oxygen Species, ROS). Other studies have associated specific forms of one of the genes encoding KGDHC (namely the DLST gene) with AD, Parkinson's disease, as well as other neurodegenerative diseases. Reductions in KGDHC activity can be plausibly linked to several aspects of brain dysfunction and neuropathology in a number of neurodegenerative diseases. Further studies are needed to assess mechanisms underlying the sensitivity of KGDHC to oxidative stress and the relation of KGDHC deficiency to selective vulnerability in neurodegenerative diseases.     

Quantitative alpha-ketoglutarate dehydrogenase activity staining in brain sections and in cultured cells

The activity of a key mitochondrial enzyme, the alpha-ketoglutarate dehydrogenase complex (KGDHC), declines in the brains of patients with neurodegenerative diseases such as Alzheimer's disease, as well as in thiamine-deficient (TD) animals. The decreased activity often occurs without a reduction in enzyme protein, which negates the use of immunocytochemistry to study cellular or regional changes in enzyme activity within the brain. To overcome this limitation, an activity staining method using nitroblue tetrazolium was developed. The histochemical activity staining was standardized in cultured cells. The assay was linear with time and was highly specific for KGDHC. The dark-blue reaction product (formazan) formed a pattern that was consistent with mitochondrial localization. Treatment of the cultured cells with both reversible and irreversible inhibitors decreased formazan production, whereas conventional enzyme assays on cell lysates only revealed loss of KGDHC activity with irreversible inhibitors. The activity staining was also linear with time and highly specific for KGDHC activity in mouse brain sections. Staining occurred throughout the brain, and discrete neuronal populations exhibited particularly intense staining. The pattern of staining differed markedly from the distribution of KGDHC protein by immunocytochemistry. Generalized decreases in the intensity of activity staining that occurred in the TD brains compared to controls were comparable with the loss of KGDHC activity by conventional enzyme assay. Thus, the present study introduces a new histochemical method to measure KGDHC activity at the cellular and regional level, which will be useful to determine changes of in situ enzyme activity.     

Brain α-Ketoglutarate Dehydrogenase Complex: Kinetic Properties, Regional Distribution, and Effects of Inhibitors

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.     

Novel functions of the alpha-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer's disease

Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H(2)O(2) and to probe the mechanism underlying these changes. Since the response to H(2)O(2) is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one-hour treatment with H(2)O(2) decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H(2)O(2) inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H(2)O(2) converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H(2)O(2) diminished glutathione to a similar level after 24 h. However, H(2)O(2), but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H(2)O(2) together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes.     

A complex effect of arsenite on the formation of alpha-ketoglutarate in rat liver mitochondria

This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial alpha-keto acid dehydrogenases. Arsenite at concentrations of 0.1-1.0 mM was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing alpha-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating alpha-ketoglutarate; however, at low concentrations of alpha-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD+ content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 mM; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mg2+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of alpha-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations.     

Measurement of alpha-ketoglutarate dehydrogenase activity in tissue extracts and human platelets using reversed-phase high-performance liquid chromatography

A new method for the determination of the activity of alpha-ketoglutarate dehydrogenase complex (KGDHC) in mouse brain and liver mitochondria and in human platelets using reversed-phase high-performance liquid chromatography is described. This method is based on the quantification of succinyl-CoA formed in the reaction catalyzed by KGDHC. Succinyl-CoA was separated using a YMC-Pack C8 column employing isocratic elution and detected spectrophotometrically at 254 nm. The detection limit of succinyl-CoA was 0.05 nmol. Succinyl-CoA in the supernatant of the assay mixture was stable for several hours at 4 degrees C and for a week when stored at -20 degrees C. The KGDHC assay showed good linearity with time and added protein, and all tissues demonstrated an absolute requirement for added alpha-ketoglutarate, nicotinamide dinucleotide, and coenzyme A and partial or no requirement for thiamine pyrophosphate, magnesium chloride, and dithiothreitol. The specific activities in liver and brain mitochondria and platelet homogenates determined by the present method were 19.2 +/- 0.9, 18.1 +/- 2.8, and 2.6 +/- 0.3 nmol/min/mg protein, respectively. In human platelets, the present method gives higher specific activity and lower blank values than a prior method using 14CO2 and may be useful in the diagnosis of KGDHC deficiency. This method is simple, rapid, and can be readily employed for the determination of KGDHC activity in various animal tissues and human platelets.     

Inhibition of the alpha-ketoglutarate dehydrogenase complex by the myeloperoxidase products, hypochlorous acid and mono-N-chloramine

Abstract alpha-Ketoglutarate dehydrogenase (KGDHC) complex activity is diminished in a number of neurodegenerative disorders and its diminution in Alzheimer Disease (AD) is thought to contribute to the major loss of cerebral energy metabolism that accompanies this disease. The loss of KGDHC activity appears to be predominantly due to post-translation modifications. Thiamine deficiency also results in decreased KGDHC activity and a selective neuronal loss. Recently, myeloperoxidase has been identified in the activated microglia of brains from AD patients and thiamine-deficient animals. Myeloperoxidase produces a powerful oxidant, hypochlorous acid that reacts with amines to form chloramines. The aim of this study was to investigate the ability of hypochlorous acid and chloramines to inhibit the activity of KGDHC activity as a first step towards investigating the role of myeloperoxidase in AD. Hypochlorous acid and mono-N-chloramine both inhibited purified and cellular KGDHC and the order of inhibition of the purified complex was hypochlorous acid (1x) > mono-N-chloramine (approximately 50x) > hydrogen peroxide (approximately 1,500). The inhibition of cellular KGDHC occurred with no significant loss of cellular viability at all exposure times that were examined. Thus, hypochlorous acid and chloramines have the potential to inactivate a major target in neurodegeneration.     

Reduction in the E2k subunit of the alpha-ketoglutarate dehydrogenase complex has effects independent of complex activity

The activity of the alpha-ketoglutarate dehydrogenase complex (KGDHC) declines in brains of patients with several neurodegenerative diseases. KGDHC consists of multiple copies of E1k, E2k, and E3. E1k and E2k are unique to KGDHC and may have functions independent of the complex. The present study tested the consequences of different levels of diminished E2k mRNA on protein levels of the subunits, KGDHC activity, and physiological responses. Human embryonic kidney cells were stably transfected with an E2k sense or antisense expression vector. Sense control (E2k-mRNA-100) was compared with two clones in which the mRNA was reduced to 67% of control (E2k-mRNA-67) or to 30% of control (E2k-mRNA-30). The levels of the E2k protein in clones paralleled the reduction in mRNA, and E3 proteins were unaltered. Unexpectedly, the clone with the greatest reduction in E2k protein (E2k-mRNA-30) had a 40% increase in E1k protein. The activity of the complex was only 52% of normal in E2k-mRNA-67 clone, but was near normal (90%) in E2k-mRNA-30 clone. Subsequent experiments tested whether the physiological consequences of a reduction in E2k mRNA correlated more closely to E2k protein or to KGDHC activity. Growth rate, increased DCF-detectable reactive oxygen species, and cell death in response to added oxidant were proportional to E2k proteins, but not complex activity. These results were not predicted because subunits unique to KGDHC have never been manipulated in mammalian cells. These results suggest that in addition to its essential role in metabolism, the E2k component of KGDHC may have other novel roles.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

Inhibition of pigeon breast muscle alpha-ketoglutarate dehydrogenase by phosphonate analogues of alpha-ketoglutarate.

Succinylphosphonate (SP) is a powerful inhibitor of alpha-ketoglutarate dehydrogenase (KGD). Methylation of the phosphonate reduces its inhibitory effect. The complex of KGD with SP undergoes a kinetically slow transition similar to the process observed during catalysis. alpha-Ketoglutarate binds to the enzyme-inhibitor complex, preventing its isomerisation.     

Inhibition of the alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular calcium regulation

Mitochondrial dysfunction occurs in many neurodegenerative diseases. The alpha-ketoglutarate dehydrogenase complex (KGDHC) catalyzes a key and arguably rate-limiting step of the tricarboxylic acid cycle (TCA). A reduction in the activity of the KGDHC occurs in brains and cells of patients with many of these disorders and may underlie the abnormal mitochondrial function. Abnormalities in calcium homeostasis also occur in fibroblasts from Alzheimer's disease (AD) patients and in cells bearing mutations that lead to AD. Thus, the present studies test whether the reduction of KGDHC activity can lead to the alterations in mitochondrial function and calcium homeostasis. alpha-Keto-beta-methyl-n-valeric acid (KMV) inhibits KGDHC activity in living N2a cells in a dose- and time-dependent manner. Surprisingly, concentration of KMV that inhibit in situ KGDHC by 80% does not alter the mitochondrial membrane potential (MMP). However, similar concentrations of KMV induce the release of cytochrome c from mitochondria into the cytosol, reduce basal [Ca(2+)](i) by 23% (P<0.005), and diminish the bradykinin (BK)-induced calcium release from the endoplasmic reticulum (ER) by 46% (P<0.005). This result suggests that diminished KGDHC activities do not lead to the Ca(2+) abnormalities in fibroblasts from AD patients or cells bearing PS-1 mutations. The increased release of cytochrome c with diminished KGDHC activities will be expected to activate other pathways including cell death cascades. Reductions in this key mitochondrial enzyme will likely make the cells more vulnerable to metabolic insults that promote cell death.     

Analysis of conformationally restricted alpha-ketoglutarate analogues as substrates of dehydrogenases and aminotransferases.

Five synthetic, conformationally restricted alpha-ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransferases. The compounds were found not to be detectable substrates of glutamate dehydrogenase, L-leucine dehydrogenase, L-phenylalanine dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartate aminotransferase, alanine aminotransferase, and alpha-ketoglutarate dehydrogenase complex. However, two thermostable aminotransferases were identified that catalyze transamination between several L-amino acids (e.g., phenylalanine, glutamate) and the alpha-ketoglutarate analogues of interest. Transamination between L-glutamate (or L-phenylalanine) and the alpha-ketoglutarate analogues was found to be 0.13 to 1.08 micromol/h/mg at 45 degrees C. The products resulting from transamination between L-phenylalanine and the alpha-ketoglutarate analogues were separated by reverse-phase HPLC, and the newly formed amino acid analogues were analyzed by LC-MS in an ion selective mode. In each case, the ions obtained were consistent with the expected product and a representative example is provided. The possibility existed that although the alpha-ketoglutarate analogues are not substrates of the dehydrogenases and most of the aminotransferases investigated, they might be good inhibitors. Weak inhibition of aminotransferases and glutamate dehydrogenase was found with some of the alpha-ketoglutarate analogues. The newly available thermostable aminotransferases may have general utility in the synthesis of bulky L-amino acids from the corresponding alpha-keto acids.     

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.     

Functional role of histidine residues of alpha-ketoglutarate dehydrogenase

The protective effect of alpha-ketoglutarate dehydrogenase substrate and its analogs on the enzyme inactivation by diethylpyrocarbonate was studied. The values of true rate constants for diethylpyrocarbonate-induced inactivation and the Kd values for the enzyme complexes with ligands were determined. A comparison of Kd values for a number of ligands suggests that the histidine residue of the enzyme active center interacts with the alpha-keto group of the substrate. A mechanism of this histidine residue involvement in the catalytic act is proposed. According to this mechanism, the imidazole ring of histidine which is responsible for the substrate activation causes a simultaneous formation of a catalytically active form of the coenzyme--thiamine pyrophosphate ilide. It is assumed that the lower (as compared with the enzyme-substrate complexes) values of rate constants of inactivation by diethylpyrocarbonate for alpha-ketoglutarate dehydrogenase complexes with succinate, glutarate, and oxaloacetate are due to additional protonation of the histidine residue, eventually resulting in the blocking of the analogs interaction with the coenzyme.     

Inactivation and reactivation of the mitochondrial α-ketoglutarate dehydrogenase complex

Reduced brain metabolism is an invariant feature of Alzheimer Disease (AD) that is highly correlated to the decline in brain functions. Decreased activities of key tricarboxylic acid cycle (TCA) cycle enzymes may underlie this abnormality and are highly correlated to the clinical state of the patient. The activity of the α-ketoglutarate dehydrogenase complex (KGDHC), an arguably rate-limiting enzyme of the TCA cycle, declines with AD, but the mechanism of inactivation and whether it can be reversed remains unknown. KGDHC consists of multiple copies of three subunits. KGDHC is sensitive to oxidative stress, which is pervasive in AD brain. The present studies tested the mechanism for the peroxynitrite-induced inactivation and subsequent reactivation of purified and cellular KGDHC. Peroxynitrite inhibited purified KGDHC activity in a dose-dependent manner and reduced subunit immunoreactivity and increased nitrotyrosine immunoreactivity. Nano-LC-MS/MS showed that the inactivation was related to nitration of specific tyrosine residues in the three subunits. GSH diminished the nitrotyrosine immunoreactivity of peroxynitrite-treated KGDHC, restored the activity and the immunoreactivity for KGDHC. Nano-LC-MS/MS showed this was related to de-nitration of specific tyrosine residues, suggesting KGDHC may have a denitrase activity. Treatment of N2a cells with peroxynitrite for 5 min followed by recovery of cells for 24 h reduced KGDHC activity and increased nitrotyrosine immunoreactivity. Increasing cellular GSH in peroxynitrite-treated cells rescued KGDHC activity to the control level. The results suggest that restoring KGDHC activity is possible and may be a useful therapeutic approach in neurodegenerative diseases.     

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.     

Cosubstrate binding site of Pseudomonas sp. AK1 gamma-butyrobetaine hydroxylase. Interactions with structural analogs of alpha-ketoglutarate

Forty-one aromatic and aliphatic analogs of alpha-ketoglutarate were studied kinetically for their interaction with the alpha-ketoglutarate binding site of gamma-butyrobetaine hydroxylase obtained from Pseudomonas sp. AK1. Together, the compounds represent structural permutations probing the contribution of: 1) the C5 carboxyl group of alpha-ketoglutarate (domain I); 2) the C1-C2 keto acid moiety of alpha-ketoglutarate (domain II); 3) the distance between domains I and II; and 4) the spatial relationship of the two domains required for optimal interaction with the cosubstrate binding site. All compounds were competitive inhibitors for alpha-ketoglutarate (Km 0.018 mM). Functionally, two subsites of the cosubstrate binding site were evident: subsite I for polar interaction with the C5 carboxyl group, and subsite II, comprising of two distinct cis-oriented coordination sites of the catalytic ferrous ion which interact with the C1-C2 keto acid moiety. The most efficient inhibitors were pyridine 2,4-dicarboxylate (Ki 0.0002 mM) and 3,4-dihydroxybenzoate (Ki 0.0006 mM). Both compounds contain a carboxyl group and a chelating moiety corresponding to domains I and II of alpha-ketoglutarate, respectively. The fixed orientation of these groups in both analogs was used to assess intersubsite distance and spatial relationship required for optimal interaction with the cosubstrate binding site. Binding at subsite I and chelation at subsite II were indispensible for effective competitive inhibition. The distance between these two domains also helped determine whether attachment at the cosubstrate binding site would be catalytically productive. This was emphasized by the failure of either oxaloacetate or alpha-ketoadipinate to promote hydroxylation. Optimal interdomain distance, however, was not sufficient for cosubstrate utilization, as pyridine 2,4-dicarboxylate, with an interdomain distance identical to alpha-ketoglutarate in its staggered conformation, did not sustain hydroxylation. In the overall, these studies suggest that alpha-ketoglutarate utilization occurs in a ligand reaction at the active site ferrous ion of gamma-butyrobetaine hydroxylase. This is of particular interest since the delineated stereochemical mode of oxidative decarboxylation could generate the reactive oxo-iron species that was shown experimentally to promote gamma-butyrobetaine hydroxylation by an abstraction-recombination mechanism (Blanchard, J. S., and Englard, S. (1983) Biochemistry 22, 5922-5928; Englard, S., Blanchard, J. S., and Midelfort, C. F. (1985) Biochemistry 24, 1110-1116).     

Cisplatin-induced toxicity is associated with platinum deposition in mouse kidney mitochondria in vivo and with selective inactivation of the alpha-ketoglutarate dehydrogenase complex in LLC-PK1 cells

The anticancer drug cisplatin is nephrotoxic and neurotoxic. Previous data support the hypothesis that cisplatin is bioactivated to a nephrotoxicant. The final step in the proposed bioactivation is the formation of a platinum-cysteine S-conjugate followed by a pyridoxal 5'-phosphate (PLP)-dependent cysteine S-conjugate beta-lyase reaction. This reaction would generate pyruvate, ammonium, and a highly reactive platinum (Pt)-thiol compound in vivo that would bind to proteins. In this work, the cellular location and identity of the PLP-dependent cysteine S-conjugate beta-lyase were investigated. Pt was shown to bind to proteins in kidneys of cisplatin-treated mice. The concentration of Pt-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Treatment of the mice with aminooxyacetic acid (AOAA, a PLP enzyme inhibitor), which had previously been shown to block the nephrotoxicity of cisplatin, decreased the binding of Pt to mitochondrial proteins but had no effect on the amount of Pt bound to proteins in the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLP-dependent cysteine S-conjugate beta-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes beta-elimination reactions with cysteine S-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a beta-lyase reaction with the cisplatin-cysteine S-conjugate. In this study, mitAspAT was stably overexpressed in LLC-PK(1) cells. Cisplatin was significantly more toxic in confluent monolayers of LLC-PK(1) cells that overexpressed mitAspAT than in control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspAT-transfected cells. The Pt-thiol compound could rapidly bind proteins and inactivate enzymes in close proximity of the PLP-dependent cysteine S-conjugate beta-lyase. Treatment with 50 or 100 microM cisplatin for 3 h, followed by removal of cisplatin from the medium for 24 h, resulted in a pronounced loss of alpha-ketoglutarate dehydrogenase complex (KGDHC) activity in both mitAspAT-transfected cells and control cells. Exposure to 100 microM cisplatin resulted in a significantly greater loss of KGDHC activity in the cells overexpressing mitAspAT than in control cells. Aconitase activity was diminished in both cell types, but only at the higher level of exposure to cisplatin. AspAT activity was also significantly decreased by cisplatin treatment. By contrast, several other enzymes (both cytosolic and mitochondrial) involved in energy/amino acid metabolism were not significantly affected by cisplatin treatment in the LLC-PK(1) cells, whether or not mitAspAT was overexpressed. The susceptibility of KGDHC and aconitase to inactivation in kidney cells exposed to cisplatin metabolites may be due to the proximity of mitAspAT to KGDHC and aconitase in mitochondria. These findings support the hypothesis that a mitochondrial cysteine S-conjugate beta-lyase converts the cisplatin-cysteine S-conjugate to a toxicant, and the data are consistent with the hypothesis that mitAspAT plays a role in the bioactivation of cisplatin.     

The effect of acylcarnitines on the oxidation of branched chain alpha-keto acids in mitochondria

The oxidation of 14C-labelled branched-chain alpha-keto acids corresponding to the branched-chain amino acids valine, isoleucine and leucine has been studied in isolated mitochondria from heart, liver and skeletal muscle. 1. Heart and liver mitochondria have similar capacities to oxidize these alpha-keto acids based on protein content. Skeletal muscle mitochondria also show significant activity. 2. Half maximum rates are obtained with approximately 0.1 mM of the alpha-keto acids under optimal conditions. Added NAD and CoA had no effect on the oxidation rate, showing that endogenous mitochondrial NAD and CoA are required for the oxidation. 3. Addition of carnitine esters of fatty acids (C6--C16), succinate, pyruvate, or alpha-ketoglutarate inhibited the oxidation of the branched chain alpha-keto acids, especially in a high-energy state (no ADP added). In heart mitochondria the addition of AD (low-energy state) decreased the inhibitory effects of acylcarnitines of medium chain length or of pyruvate, and abolished the inhibitory effect of succinate. It is suggested that the oxidation rate is regulated mainly by the redox state of the mitochondria under the conditions used. 4. The results are discussed in relation to the regulation of branched-chain amino acid metabolism in the body.