Activity Of Alpha-ketoglutarate Dehydrogenase Complex Research Articles

Impact of short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs onmitochondrial energy metabolism

Accumulation of organic acids as well as their CoA and carnitine esters in tissues and body fluids is a common finding in organic acidurias, beta-oxidation defects, Reye syndrome, and Jamaican vomiting sickness. Pathomechanistic approaches for these disorders have been often focused on the effect of accumulating organic acids on mitochondrial energy metabolism, whereas little is known about the pathophysiologic role of short- and medium-chain acyl-CoAs and acylcarnitines. Therefore, we investigated the impact of short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs on central components of mitochondrial energy metabolism, namely alpha-ketoglutarate dehydrogenase complex, pyruvate dehydrogenase complex, and single enzyme complexes I-V of respiratory chain. Although at varying degree, all acyl-CoAs had an inhibitory effect on pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity. Effect sizes were critically dependent on chain length and number of functional groups. Unexpectedly, octanoyl-CoA was shown to inhibit complex III. The inhibition was noncompetitive regarding reduced ubiquinone and uncompetitive regarding cytochrome c. In addition, octanoyl-CoA caused a blue shift in the gamma band of the absorption spectrum of reduced complex III. This effect may play a role in the pathogenesis of medium-chain and multiple acyl-CoA dehydrogenase deficiency, Reye syndrome, and Jamaican vomiting sickness which are inherited and acquired conditions of intracellular accumulation of octanoyl-CoA.

Dactylorhin B reduces toxic effects of β-amyloid fragment (25–35) on neuron cells and isolated rat brain mitochondria

beta-amyloid is strongly implicated in Alzheimer's pathology, and mitochondria play an important role in neurodegenerative disorders. Dactylorhin B [short for bis(4-beta-D-glucopyranosyloxybenzyl)-2-beta-D-glucopyranosyl-2-isobutyltartrate (DHB)] is an active compound isolated from Coeloglossum viride. (L.) Hartm. var. bracteatum (Willd.) and may have neuroprotective effects. In the present study, we investigated the damage of rat brain mitochondrial function induced by beta-amyloid and the protective effect of DHB. Isolated rat brain mitochondria were incubated with amyloid-beta peptide (Abeta)(25-35) alone or together with DHB. reactive oxygen species production, pyruvate dehydrogenase complex activity, alpha-ketoglutarate dehydrogenase complex activity, cytochrome c oxidase activity, mitochondrial swelling, mitochondrial membrane potential and the release of cytochrome c from mitochondria were measured. Data showed that Abeta(25-35) directly disrupted mitochondrial function, inhibited the key enzymes and contributed to apoptosis and the deficiency of energy metabolism. Co-incubation of DHB attenuated Abeta(25-35)-induced pathological changes. The significance of DHB in the management of mitochondria-related disorders is discussed.

Thiamine-Responsive Congenital Lactic Acidosis: Clinical and Biochemical Studies

We studied six infants with thiamine-responsive congenital lactic acidosis and normal pyruvate dehydrogenase complex activity in vitro, through clinical and biochemical analysis. In addition to elevated lactate and pyruvate levels, the data revealed increased urinary excretion of alpha-ketoglutarate, alpha-ketoadipate, and branched chain ketoacids, indicating functional impairment of thiamine-requiring enzymes, such as pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, alpha-ketoadipate dehydrogenase, and branched chain amino acid dehydrogenase. The metabolism of thiamine has not been investigated in patients with thiamine-responsive congenital lactic acidosis. We evaluated two specific transport systems, THTR-1 (SLC19A2) and THTR-2 (SLC19A3), and a pyrophosphorylating enzyme of thiamine, thiamine pyrophosphokinase (hTPK 1), in addition to pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity; no abnormality was found. Although the clinical features of thiamine-responsive congenital lactic acidosis are heterogeneous and clinical responses to thiamine administration vary, we emphasize the importance of early diagnosis and initiation of thiamine therapy before the occurrence of permanent brain damage. Careful monitoring of lactate and pyruvate would be useful in determining thiamine dosage.

Bioenergetics in Glutaryl-Coenzyme A Dehydrogenase Deficiency: A ROLE FOR GLUTARYL-COENZYME A

Inherited deficiency of glutaryl-CoA dehydrogenase results in an accumulation of glutaryl-CoA, glutaric, and 3-hydroxyglutaric acids. If untreated, most patients suffer an acute encephalopathic crisis and, subsequently, acute striatal damage being precipitated by febrile infectious diseases during a vulnerable period of brain development (age 3 and 36 months). It has been suggested before that some of these organic acids may induce excitotoxic cell damage, however, the relevance of bioenergetic impairment is not yet understood. The major aim of our study was to investigate respiratory chain, tricarboxylic acid cycle, and fatty acid oxidation in this disease using purified single enzymes and tissue homogenates from Gcdh-deficient and wild-type mice. In purified enzymes, glutaryl-CoA but not glutaric or 3-hydroxyglutaric induced an uncompetitive inhibition of alpha-ketoglutarate dehydrogenase complex activity. Notably, reduced activity of alpha-ketoglutarate dehydrogenase activity has recently been demonstrated in other neurodegenerative diseases, such as Alzheimer, Parkinson, and Huntington diseases. In contrast to alpha-ketoglutarate dehydrogenase complex, no direct inhibition of glutaryl-CoA, glutaric acid, and 3-hydroxyglutaric acid was found in other enzymes tested. In Gcdh-deficient mice, respiratory chain and tricarboxylic acid activities remained widely unaffected, virtually excluding regulatory changes in these enzymes. However, hepatic activity of very long-chain acyl-CoA dehydrogenase was decreased and concentrations of long-chain acylcarnitines increased in the bile of these mice, which suggested disturbed oxidation of long-chain fatty acids. In conclusion, our results demonstrate that bioenergetic impairment may play an important role in the pathomechanisms underlying neurodegenerative changes in glutaryl-CoA dehydrogenase deficiency.

Independent modulation of the activity of alpha-ketoglutarate dehydrogenase complex by Ca2+ and Mg2+.

The activity of alpha-ketoglutarate dehydrogenase complex (KGDHC), an important enzyme regulating several metabolic pathways, could be regulated by changes in the environment within the mitochondrial matrix. It has been postulated that the activity of this and other dehydrogenases in vivo could be modulated by changes in the intramitochondrial concentrations of Ca2+ or Mg2+. Using a purified alpha-ketoglutarate dehydrogenase from pig hearts, the effect of Ca2+ and/or Mg2+ on the enzyme activity was investigated. Either Ca2+ or Mg2+ increased enzyme activity, and the effects were additive if the concentrations of free divalent cations were below 0.1 and 1 mM for Ca2+ and Mg2+, respectively. In the presence of 1 mM alpha-ketoglutarate and other cofactors, the KM for Mg2+ was 25 microM and less than 1 microM for Ca2+. The KM for alpha-ketoglutarate was a function of the divalent cation(s) present: 4 +/- 1.1 mM in the absence of Ca2+, with or without Mg2+; 2.2 mM in the presence of 1.8 microM Ca2+ alone; and 0.3 mM in the presence of both Ca2+ and Mg2+. Mg2+ increased KGDHC activity only in the presence of thiamine pyrophosphate (TPP) indicating that KGDHC requires both TPP and Mg2+ for enzyme's maximal activity. The affinity of KGDHC for NAD+ is significantly changed by either Mg2+ or Ca2+. The conclusions are that changes in both Ca2+ and Mg2+, in concentrations possibly occurring within mitochondria, could control KGDHC activity and that thiamine pyrophosphate is required for maximal enzyme activity.

Cerebellar alpha-ketoglutarate dehydrogenase activity is reduced in spinocerebellar ataxia type 1.

We measured the activity of the thiamine pyrophosphate-dependent enzyme alpha-ketoglutarate dehydrogenase complex in postmortem brain of 12 patients with the spinocerebellar ataxia type 1 form of olivopontocerebellar atrophy. alpha-Ketoglutarate dehydrogenase complex activity measured in the absence of thiamine pyrophosphate was markedly reduced (-72%) in olivopontocerebellar atrophy cerebellar cortex. Decreased activity of this key rate-limiting Krebs cycle enzyme could compromise cerebellar energy metabolism and excitatory amino acid synthesis and thereby contribute to the brain dysfunction of olivopontocerebellar atrophy.

Inhibition of α-ketoglutarate dehydrogenase activity by a distinct population of autoantibodies recognizing dihydrolipoamide succinyltransferase in primary biliary cirrhosis

Sera from patients with primary biliary cirrhosis contain autoantibodies that recognize mitochondrial proteins. Five of the target autoantigens have now been identified as enzymes of three related multienzyme complexes: the pyruvate dehydrogenase complex, the branched chain alpha-ketoacid dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex. Each complex consists of component enzymes designated E1, E2 and E3. In this report, we confirm that primary biliary cirrhosis sera react with dihydrolipoamide succinyltransferase, the E2 component of alpha-ketoglutarate dehydrogenase complex. Seventy-three of 188 (39%) primary biliary cirrhosis sera reacted with alpha-ketoglutarate dehydrogenase complex-E2 when immunoblotted against purified alpha-ketoglutarate dehydrogenase complex; one of these sera also reacted with the E1 component. In addition, primary biliary cirrhosis sera possessing alpha-ketoglutarate dehydrogenase complex-E2 reactivity specifically inhibited enzyme function of alpha-ketoglutarate dehydrogenase complex. Enzyme activity was not affected by primary biliary cirrhosis sera that contained autoantibodies to pyruvate dehydrogenase complex-E2 and/or branched chain alpha-ketoacid dehydrogenase complex-E2, which lacked alpha-ketoglutarate dehydrogenase complex-E2 reactivity. Furthermore, affinity-purified primary biliary cirrhosis sera against alpha-ketoglutarate dehydrogenase complex-E2 inhibited only alpha-ketoglutarate dehydrogenase complex activity but did not alter enzyme activity of either pyruvate dehydrogenase complex or branched chain alpha-ketoacid dehydrogenase complex. Finally, alpha-ketoglutarate dehydrogenase complex-E2 specific affinity-purified antisera did not react on immunoblot with any component enzymes of pyruvate dehydrogenase complex or branched chain alpha-ketoacid dehydrogenase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Activities of oxidative enzymes in mycoplasmas.

The activities of several oxidoreductases were measured in three fermentative and two nonfermentative Mycoplasma species that were grown under aerobic or anaerobic conditions. Acholeplasma laidlawii MG, Mycoplasma hyorhinis GDL, and Mycoplasma pneumoniae FH had very high apparent activities of pyruvate dehydrogenase and pyruvate dehydrogenase complex compared with the activities of mammalian fibroblasts or human platelet-enriched preparations, while Mycoplasma salivarium VV and Mycoplasma arthritidis 07 had very low apparent activities of these two enzymes. Strictly anaerobic growth diminished both enzymatic activities. The activity of alpha-ketoglutarate dehydrogenase complex was minimal in all five mycoplasmas that were grown under aerobic conditions, anaerobic conditions, or both. All the mycoplasmas that were examined exhibited lactate dehydrogenase and NADH-dichlorophenol indophenol oxidoreductase activities. The properties of mycoplasmal pyruvate dehydrogenase complex suggest that it differs from the mammalian enzyme.

Genetics of the alpha-ketoglutarate dehydrogenase complex of Bacillus subtilis

The enzymatic defects in a number of Bacillus subtilis mutants of the alpha-ketoglutarate dehydrogenase complex lacking activity have been investigated. Mutants in the citK locus, as well as a series of deletions of unknown length covering the citK locus, are deficient in E1 of the complex, alpha-ketoglutarate dehydrogenase, but have normal activities of E2, dehydrolipoyl transsuccinylase, and E3, lipoamide dehydrogenase. The citK mutants and the citL22 mutant show in vitro complementation of alpha-ketoglutarate dehydrogenase complex activity. The citL22 mutant is severely deficient in lipoamide dehydrogenase activity, and, as a result, lacks activity for both the alpha-ketoglutarate and the pyruvate dehydrogenase complexes. Thus, the E3 components of both complexes are identical. The citL22 mutation maps between ura and metC on the chromosome.