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PubMed2002

Inhibition of the alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase complexes by a putative aberrant metabolite of serotonin, tryptamine-4,5-dione.

A transient energy impairment with resultant release and subsequent reuptake of 5-hydroxytryptamine (5-HT) and NMDA receptor activation with consequent cytoplasmic superoxide (O(2)(-)(*)), nitric oxide (NO(*)), and peroxynitrite (ONOO(-)) generation have all been implicated in a neurotoxic cascade which ultimately leads to the degeneration of serotonergic neurons evoked by methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA). Such observations raise the possibility that the O(2)(-)(*)/NO(*)/ONOO(-)-mediated oxidation of 5-HT, as it returns via the plasma membrane transporter to the cytoplasm of serotonergic neurons when the MA/MDMA-induced energy impairment begins to subside, may generate an endogenous neurotoxin. In vitro the O(2)(-)(*)/NO(*)/ONOO(-)-mediated oxidation of 5-HT forms tryptamine-4,5-dione (T-4,5-D). When incubated with intact rat brain mitochondria, T-4,5-D strongly inhibits state 3 respiration with pyruvate or alpha-ketoglutarate as substrates at concentrations which do not affect succinate-supported (complex II) respiration. Experiments with freeze-thawed rat brain mitochondria reveal that T-4,5-D inhibits the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes. These and other properties of T-4,5-D raise the possibility that it may be an endogenously formed intraneuronal metabolite of 5-HT that contributes to the serotonergic neurotoxicity of MA and MDMA.

Animals↗
PubMed1991

Pyruvate dehydrogenase and pyruvate carboxylase. Sites of pretranslational regulation by glucose of glucose-induced insulin release in pancreatic islets.

It has been shown previously that glucose-induced insulin release is completely absent in rat pancreatic islets that had been cultured for 1 day at low glucose (1 mM) and that it is restored by culturing islets for a 2nd day at high (20 mM) glucose (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1991) Am. J. Physiol. 259, E548-E554). It has been suggested that the incapacitation of glucose's insulinotropism is due to down-regulation of the synthesis of enzymes that process glucose's metabolic signal for insulin release. In the current study, results of metabolic, enzymic, and molecular biologic experiments were each consistent with (an) intramitochondrial site(s) of down-regulation in islets cultured at low glucose. Glucose metabolism was inhibited 80% in islets cultured at 1 mM glucose. The suppression of release of 14CO2 from [6-14C]glucose greater than from [U-14C]glucose greater than [3,4-14C]glucose greater than from [1-14C]glucose in islets cultured at low glucose indicated a mitochondrial site of down-regulation because C-6 of glucose can only be converted to CO2 in the citric acid cycle, whereas C-1 can be released as CO2 in the 6-phosphogluconate dehydrogenase [corrected] reaction, and C-6 of glucose dwells in the citric acid cycle longer than carbons 2-5 of glucose. Since carbons 3 and 4 of glucose can be decarboxylated in the pyruvate dehydrogenase reaction, incomplete suppression of CO2 formation from these carbons is consistent with suppression of pyruvate carboxylation as well as decarboxylation. Formation of 3HOH from [5-3H]glucose was equal in the two groups of islets, indicating that glycolysis as far as phosphoenolpyruvate was intact. This idea was supported by assays which showed that activities of enzymes of the glycolytic pathway between glucokinase/hexokinase and pyruvate kinase were equal in both types of islets. Additional studies indicated that regulation by glucose was at transcription of genes coding for some mitochondrial enzymes. Glucokinase, malic enzyme, and fumarase mRNAs were not affected by glucose, whereas the pyruvate dehydrogenase E1 alpha subunit and pyruvate carboxylase mRNAs were decreased 85-90% in islets cultured at 1 mM glucose. Pyruvate dehydrogenase enzyme activity was decreased to a similar extent in these islets. About 24 h was required for maximal (de)induction of pyruvate dehydrogenase E1 alpha and pyruvate carboxylase mRNAs, and the amounts of transcripts were proportional to the concentrations of glucose between 1 and 20 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

A model for the spatial location of pyruvate dehydrogenase phosphatase in mammalian pyruvate dehydrogenase complex.

Recent experimental findings on the structural--functional features of pyruvate dehydrogenase phosphatase (PDP) isolated from various sources are compared. Two alternative mechanisms (a and b) of dephosphorylation of the E1 component in the pyruvate dehydrogenase complex (PDC) are discussed: a) the reaction occurs as a result of stochastic collisions of PDP and PDC, and the generation of an enzyme--substrate complex (PDP--E1--PDC) and dephosphorylation of the E1 component occur independently at different PDP binding sites on the PDC core; b) the dephosphorylation is performed simultaneously by a certain number of PDP molecules symmetrically bound on the PDC core. The second mechanism is suggested by the self-assembly theory of multicomponent enzyme systems and can be proved by kinetic experiments. Based on self-assembly principles and data on feasible binding sites of peripheral components of the PDC, the stoichiometry and mutual location of PDP, pyruvate dehydrogenase kinase, and the E1 component on the core of mammalian PDC are postulated to provide optimal functioning of the PDC. Structural mechanisms of stimulation of PDP activity by Ca2+ and polyamines are also discussed.

Animals↗
PubMed2006

Acetylphosphinate is the most potent mechanism-based substrate-like inhibitor of both the human and Escherichia coli pyruvate dehydrogenase components of the pyruvate dehydrogenase complex.

Two analogues of pyruvate, acetylphosphinate and acetylmethylphosphinate were tested as inhibitors of the E1 (pyruvate dehydrogenase) component of the human and Escherichia coli pyruvate dehydrogenase complexes. This is the first instance of such studies on the human enzyme. The acetylphosphinate is a stronger inhibitor of both enzymes (Ki < 1 microM) than acetylmethylphosphinate. Both inhibitors are found to be reversible tight-binding inhibitors. With both inhibitors and with both enzymes, the inhibition apparently takes place by formation of a C2alpha-phosphinolactylthiamin diphosphate derivative, a covalent adduct of the inhibitor and the coenzyme, mimicking the behavior of substrate and forming a stable analogue of the C2alpha-lactylthiamin diphosphate. Formation of the intermediate analogue in each case is confirmed by the appearance of a positive circular dichroism band in the 305-306 nm range, attributed to the 1',4'-iminopyrimidine tautomeric form of the coenzyme. It is further shown that the alphaHis63 residue of the human E1 has a role in the formation of C2alpha-lactylthiamin diphosphate since the alphaHis63Ala variant is only modestly inhibited by either inhibitor, nor did either compound generate the circular dichroism bands assigned to different tautomeric forms of the 4'-aminopyrimidine ring of the coenzyme seen with the wild-type enzyme. Interestingly, opposite enantiomers of the carboligase side product acetoin are produced by the human and bacterial enzymes.

Circular Dichroism↗
PubMed1992

A mutation in the E1 alpha subunit of pyruvate dehydrogenase associated with variable expression of pyruvate dehydrogenase complex deficiency.

Defects in pyruvate dehydrogenase, the first catalytic component of the pyruvate dehydrogenase complex, are the most common cause of pyruvate dehydrogenase complex deficiency. A family with variable pyruvate dehydrogenase complex deficiency had been described in which cultured skin fibroblasts of affected family members had normal pyruvate dehydrogenase complex activity, but different tissues and blood lymphocytes had significantly diminished activities. Enzymatic activity and immunoblot studies indicated that pyruvate dehydrogenase was affected. Further evidence is presented here showing that the defect affecting pyruvate dehydrogenase complex activity is posttranscriptional. Sequencing of the coding region of the alpha-subunit of pyruvate dehydrogenase revealed a point mutation in the codon for amino acid 234 resulting in a substitution of glycine for arginine. Study of other members of the family suggested that this mutation is inherited in a sex-linked mode. The point mutation is located in a highly conserved region of the pyruvate dehydrogenase alpha-subunit gene that contains both hydrophobic and positively charged amino acid residues. Variable expression of pyruvate dehydrogenase complex deficiency in this case may be due to instability of the pyruvate dehydrogenase heterotetramer in specific tissues because of a disruption in subunit-subunit interaction.

Amino Acid Sequence↗
PubMed1988

Identification of a cDNA clone for the beta-subunit of the pyruvate dehydrogenase component of human pyruvate dehydrogenase complex.

We report the isolation of a 1.5 kb cDNA clone for the beta subunit of human pyruvate dehydrogenase (E1) from a human liver lambda gt11 cDNA library using anti-E1 serum. We generated a peptide sequence of 24 amino acids starting from the N-terminus of bovine heart mature E1 beta. The identity of the E1 beta cDNA clone was confirmed by the similarity between the amino acid sequence deduced from the cDNA nucleotide sequence and the known amino acid sequence of bovine heart E1 beta. In Northern analysis of total RNA extracted from human heart, the E1 beta cDNA clone hybridized to a major 1.6 kb and a minor 5.2 kb RNA species.

Amino Acid Sequence↗
PubMed1992

Mechanisms of fructose-induced hypertriglyceridaemia in the rat. Activation of hepatic pyruvate dehydrogenase through inhibition of pyruvate dehydrogenase kinase.

1. The effects of purified diets containing 70% glucose or 70% fructose on the activation state of hepatic pyruvate dehydrogenase (PDHa), activity of mitochondrial PDH kinase, plasma triacylglycerols (TG) and hepatic lipogenesis de novo in rats were measured. 2. Plasma TG were significantly increased in the fructose-fed compared with the glucose-fed group (125 +/- 45 mg/dl versus 57 +/- 19 mg/dl; P less than 0.002) after 3-5 weeks on the diet despite less daily food intake. 3. Hepatic PDHa in fructose-fed rats was 144% of the value in glucose-fed rats (15.4 +/- 1.2% versus 10.7 +/- 0.5%; P less than 0.002), whereas cardiac muscle PDHa was not different (45.5 +/- 6.6% versus 41.0 +/- 7.8%). 4. Intrinsic hepatic PDH kinase activity was decreased to 34% of glucose-fed values by fructose feeding (-k = 3.56 +/- 0.39 versus 10.41 +/- 1.85 min-1; P less than 0.005). 5. The fractional contribution to very-low-density-lipoprotein palmitate from hepatic lipogenesis de novo, measured by a stable-isotope mass-spectrometric method, was 10.49 +/- 2.42% (n = 8) in fructose-fed rats versus 5.55 +/- 1.38% (n = 9) in glucose-fed rats (P less than 0.05), and 2.66 +/- 2.39% (n = 3) in chow-fed rats (P less than 0.05 versus fructose-fed group). The absolute contribution to circulating TG from lipogenesis de novo was also significantly higher in the fructose-fed than in the glucose-fed group (14.9 +/- 5.1 mg/dl versus 2.9 +/- 0.6 mg/dl; P less than 0.05) 6. Portal insulin concentrations were significantly higher in the fructose-fed rats (206 +/- 49 mu-units/ml versus 81 +/- 15 mu-units/ml; P less than 0.05). 7. In conclusion, dietary fructose appears to have a specific activating effect on hepatic PDH, mediated at least in part by inhibition of PDH kinase. These results are consistent with increased flux through hepatic PDH and synthesis of new fat, not just increased re-esterification of non-esterified fatty acids.

Animals↗
PubMed1982

Purification of bovine kidney and heart pyruvate dehydrogenase phosphatase on Sepharose derivatized with the pyruvate dehydrogenase complex.

Pyruvate dehydrogenase phosphatase has been purified to apparent homogeneity from mitochondrial extracts of both beef heart and beef kidney. An essential step in this three-step purification is affinity chromatography of a largely purified phosphatase fraction using Sepharose beads to which pyruvate dehydrogenase complex is covalently bound through the lipoic acid residues of the dihydrolipoyl transacetylase component of the complex. The purified phosphatase, which has a native relative molecular mass, Mr, of about 140000, is composed of two nonidentical subunits of Mr 89000 and 49000.

Animals↗
PubMed1996

Lipoyl domain-based mechanism for the integrated feedback control of the pyruvate dehydrogenase complex by enhancement of pyruvate dehydrogenase kinase activity.

To conserve carbohydrate reserves, the reaction of the pyruvate dehydrogenase complex (PDC) must be down-regulated when the citric acid cycle is provided sufficient acetyl-CoA. PDC activity is reduced primarily through increased phosphorylation of its pyruvate dehydrogenase (E1) component due to E1 kinase activity being markedly enhanced by elevated intramitochondrial NADH:NAD+ and acetyl-CoA:CoA ratios. A mechanism is evaluated in which enhanced kinase activity is facilitated by the build-up of the reduced and acetylated forms of the lipoyl moieties of the dihydrolipoyl acetyltransferase (E2) component through using NADH and acetyl-CoA in the reverse of the downstream reactions of the complex. Using a peptide substrate, kinase activity was stimulated by these products, ruling out the possibility kinase activity is increased due to changes in the reaction state of its substrate, E1 (thiamin pyrophosphate). Each E2 subunit contains two lipoyl domains, an NH2-terminal (L1) and the inward lipoyl domain (L2), which were individually produced in fully lipoylated forms by recombinant techniques. Although reduction and acetylation of the L1 domain or free lipoamide increased kinase activity, those modifications of the lipoate of the kinase-binding L2 domain gave much greater enhancements of kinase activity. The large stimulation of the kinase generated by acetyl-CoA only occurred upon addition of the transacetylase-catalyzing (lipoyl domain-free) inner core portion of E2 plus a reduced lipoate source, affirming that acetylation of this prosthetic group is an essential mechanistic step for acetyl-CoA enhancing kinase activity. Similarly, the lesser stimulation of kinase activity by just NADH required a lipoate source, supporting the need for lipoate reduction by E3 catalysis. Complete enzymatic delipoylation of PDC, the E2-kinase subcomplex, or recombinant L2 abolished the stimulatory effects of NADH and acetyl-CoA. Retention of a small portion of PDC lipoates lowered kinase activity but allowed stimulation of this residual kinase activity by these products. Reintroduction of lipoyl moieties, using lipoyl protein ligase, restored the capacity of the E2 core to support high kinase activity along with stimulation of that activity up to 3-fold by NADH and acetyl-CoA. As suggested by those results, the enhancement of kinase activity is very responsive to reductive acetylation with a half-maximal stimulation achieved with approximately 20% of free L2 acetylated and, from an analysis of previous results, with acetylation of only 3-6 of the 60 L2 domains in intact PDC. Based on these findings, we suggest that kinase stimulation results from modification of the lipoate of an L2 domain that becomes specifically engaged in binding the kinase. In conclusion, kinase activity is attenuated through a substantial range in response to modest changes in the proportion of oxidized, reduced, and acetylated lipoyl moieties of the L2 domain of E2 produced by fluctuations in the NADH:NAD+ and acetyl-CoA:CoA ratios as translated by the rapid and reversible E3 and E2 reactions.

Acetyl Coenzyme A↗
PubMed1992

Purification and partial characterization of rat liver pyruvate dehydrogenase kinase activator protein (free pyruvate dehydrogenase kinase).

Rat liver pyruvate dehydrogenase (PDH) kinase activator protein (KAP), a free PDH kinase readily separable from PDH complex and its intrinsic kinase, has been purified to apparent homogeneity from liver mitochondria of fed and 48-h starved rats. On SDS-PAGE an apparently single band of M(r) 45 kDa was obtained. N-Terminal amino acid sequence analyses (8-10 cycles) confirmed the presence of a single peptide in each case. The specific activity of the purified KAP from 48-h starved rats (14,413 U/mg protein) was 4.5-fold greater than that from fed rats.

Amino Acid Sequence↗
PubMed1997

Dihydrolipoamide dehydrogenase-binding protein of the human pyruvate dehydrogenase complex. DNA-derived amino acid sequence, expression, and reconstitution of the pyruvate dehydrogenase complex.

Protein X, recently renamed dihydrolipoamide dehydrogenase-binding protein (E3BP), is required for anchoring dihydrolipoamide dehydrogenase (E3) to the dihydrolipoamide transacetylase (E2) core of the pyruvate dehydrogenase complexes of eukaryotes. DNA and deduced protein sequences for E3BP of the human pyruvate dehydrogenase complex are reported here. With the exception of only a single lipoyl domain, the protein has a segmented multi-domain structure analogous to that of the E2 component of the complex. The protein has 46% amino acid sequence identity in its amino-terminal region with the second lipoyl domain of E2, 38% identity in its central region with the putative peripheral subunit-binding domain of E2, and 50% identity in its carboxyl-terminal region with the catalytic inner core domain of E2. The similarity in the latter domain stands in contrast to E3BP of Saccharomyces cerevisiae, which is quite different from its homologous transacetylase in this region. The putative catalytic site histidine residue present in the inner core domains of all dihydrolipoamide acyltransferases is replaced by a serine residue in human E3BP; thus, catalysis of coenzyme A acetylation by this protein is unlikely. Coexpression of cDNAs for E3BP and E2 resulted in the formation of an E2.E3BP subcomplex that spontaneously reconstituted the pyruvate dehydrogenase complex in the presence of native E3 and recombinant pyruvate decarboxylase (E1).

Amino Acid Sequence↗
PubMed1983

The substrate-mediated inactivation of the pyruvate dehydrogenase component of the pigeon breast muscle pyruvate dehydrogenase complex.

Incubation of the pyruvate dehydrogenase component isolated from the pigeon breast muscle pyruvate dehydrogenase complex with Mg2+, thiamine pyrophosphate and low concentrations of pyruvic acid in the absence of electron acceptors results in irreversible time-dependent inactivation of the enzyme. The rate of the enzyme inactivation is markedly decreased in the presence of high concentrations of pyruvate; in this case acetoin and acetolactate are detected in the reaction mixture. The enzyme activity is stabilized when the artificial electron acceptor, 2,6-dichlorophenolindophenol, is present in the reaction mixture. The substrate-mediated inactivation of the enzyme is accompanied by incorporation of the 2-[14C]-substrate fragment and labelled thiamine pyrophosphate into the protein fraction. The enzyme reactivation by neutral hydroxylamine and the protective effect of dithiothreitol suggest that the SH-group(s) may be involved in the substrate-mediated inactivation of pyruvate dehydrogenase.

Animals↗
PubMed1985

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.

Acetylation↗
PubMed1977

Fluorescent derivatives of the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex.

One sulfhydryl group per polypeptide chain of the pyruvate dehydrogenase component of the pyruvate dehydrogenase multienzyme complex from Escherichia coli was selectively labeled with N-[P-(2-benzoxazoyl)phenyl]-maleimide (NBM), 4-dimethylamino-4-magnitude of-maleimidostilbene (NSM), and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) in 0.05 M potassium phosphate (pH 7). Modification of the sulfhydryl group did not alter the enzymatic activity or the binding of 8-anilino-1-naphthalenesulfonate (ANS) or thiochrome diphosphate to the enzyme. The fluorescence of the NBM or NSM coupled to the sulfhydryl group on the enzyme was quenched by binding to the enzyme of the substrate pyruvate the coenzyme thiamine diphosphate, the coenzyme analogue thiochrome diphosphate, the regulatory ligands acetyl-CoA, GTP, and phosphoenolpyruvate, and the acetyl-CoA analogue, ANS. Fluorescence energy transfer measurements were carried out for the enzyme-bound donor-acceptor pairs NBM-ANS, NBM-thiochrome diphosphate ANS-DDPM, and thiochrome diphosphate-DDM. The results indicate that the modified sulfhydryl group is more than 40 A from the active site and approximately 49 A from the acetyl-CoA regulatory site. Thus, a conformational change must accompany the binding of ligands to the regulatory and catalytic sites. Anisotropy depolarization measurements with ANS bound on the isolated pyruvate dehydrogenase in 0.05 M potassium phosphate (pH 7.0) suggest that under these conditions the enzyme is dimeric.

Anilino Naphthalenesulfonates↗