Plant pyruvate dehydrogenase complex purification, characterization and regulation by metabolites and phosphorylation

Randall, Douglas D.; Rubin, Paul M.; Fenko, Michael D.

doi:10.1016/0005-2744(77)90169-3

Cited by 58 publications

(25 citation statements)

References 32 publications

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“…In contrast, PDC and KGDC reactions tolerate high Triton X-100 concentrations (Randall et al, 1977;Dry and Wiskich, 1987). Provision of exogenous pig E3 to the activity assay in the presence of Triton X-100 did not recover BCKDC activity, suggesting that the lability was not simply caused by loss of E3.…”

Section: Identification Of Lipoic Acid-containing Proteins In Plantmentioning

confidence: 97%

Lipoic Acid-Dependent Oxidative Catabolism of α-Keto Acids in Mitochondria Provides Evidence for Branched-Chain Amino Acid Catabolism in Arabidopsis

et al. 2004

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Lipoic acid-dependent pathways of ␣-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/ sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and ␣-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD ϩ was analyzed by spectrophotometry using specific ␣-keto acids. Pyruvate-and ␣-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the ␣-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid-and ␣-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived ␣-keto acid, ␣-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.The enzymatic decarboxylation of ␣-keto organic acids in cells is often facilitated by the cofactor thiamine pyrophosphate (TPP), which acts as a strong nucleophile to attack the carbonyl carbon of ␣-keto acids. The remaining aldehyde can be simply protonated; for example, during acetaldehyde formation by pyruvate decarboxylase (EC 4.1.1.1) or, alternatively, the energy released by the decarboxylation can be coupled to the generation of NADH. The ␣-keto acid decarboxylating dehydrogenase complexes encapsulate this alternative reaction series. These complex structures use E1 (␣-keto acid dehydrogenase), E2 (acyltransferase), and E3 (lipoamide dehydrogenase) enzymes and five cofactors (TPP, CoA, lipoic acid, FAD ϩ , and NAD ϩ ) in their catalytic cycle (Reed, 1974;Mooney et al., 2002). Active-site coupling is used to catalyze decarboxylation of ␣-keto acids (E1 enzyme), esterification of aldehydes to CoA (E2 enzyme), and reduction of NAD ϩ to NADH (E3 enzyme; Reed, 1981;Perham et al., 2002). The most widely studied ␣-keto acid enzyme complexes are the pyruvate dehydrogenase complex (PDC) that regulates the entry of carbon into the tricarboxylic acid (TCA) cycle by the provision of acetyl-CoA and the ␣-ketoglutarate dehydrogenase complex (KGDC) that acts within the TCA cycle to synthesize succinylCoA. B...

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Section: Identification Of Lipoic Acid-containing Proteins In Plantmentioning

confidence: 97%

Lipoic Acid-Dependent Oxidative Catabolism of α-Keto Acids in Mitochondria Provides Evidence for Branched-Chain Amino Acid Catabolism in Arabidopsis

et al. 2004

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“…In the latter case we inactivated the endogenous mitochondrial PDCs by addition of ATP. In the presence of ATP an endogenous PDC kinase converts the plant and fungal mitochondrial PDCs to their phosphorylated and inactive forms (33,34) (the kinase has no effect on E. coli PDC). The N. crassa transferase was also active with octanoyl-ACP.…”

Section: Acp-dependent Activation Of Pyruvate Dehydrogenase Complex 1mentioning

confidence: 99%

A New Metabolic Link

Jordan¹,

Cronan²

1997

Journal of Biological Chemistry

144

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Lipoic acid is an essential enzyme cofactor that requires covalent attachment to its cognate proteins to confer biological activity. The major lipoylated proteins are highly conserved enzymes of central metabolism, the pyruvate and ␣-ketoglutarate dehydrogenase complexes. The classical lipoate ligase uses ATP to activate the lipoate carboxyl group followed by attachment of the cofactor to a specific subunit of each dehydrogenase complex, and it was assumed that all lipoate attachment proceeded by this mechanism. However, our previous work indicated that Escherichia coli could form lipoylated proteins in the absence of detectable ATP-dependent ligase activity raising the possibility of a class of enzyme that attaches lipoate to the dehydrogenase complexes by a different mechanism. We now report that E. coli and mitochondria contain lipoate transferases that use lipoyl-acyl carrier protein as the lipoate donor. This finding demonstrates a direct link between fatty acid synthesis and lipoate attachment and also provides the first direct demonstration of a role for the enigmatic acyl carrier proteins of mitochondria.Lipoic acid is a cofactor required for function of the citric acid cycle (1). Two key enzyme multienzyme complexes use covalently bound lipoic acid to carry reaction intermediates between the active sites of the complexes. These enzymes are the pyruvate dehydrogenase complex (PDC) 1 that is responsible for the synthesis of the acetyl-CoA needed to prime the cycle and the ␣-ketoglutarate dehydrogenase complex (KGDC) that catalyzes the C 5 to C 4 step of the cycle (2). Covalently attached lipoic acid not only provides substrate channeling between the different active sites of these complexes but also maintains the activated carboxyl group in thioester linkage for delivery to coenzyme A. Covalent attachment of lipoic acid to the E 2 subunits of these enzyme complexes is required for activity in vivo. The cofactor is attached via an amide linkage formed between the lipoate carboxyl group and the ⑀-amino group of a specific lysine residue of a well conserved E 2 subunit N-terminal domain (2). The first enzyme shown to attach lipoic acid to the lipoyl domains of PDC catalyzes a two-step reaction in which ATP activates lipoic acid to lipoyl-AMP followed by transfer of the lipoyl moiety to the appropriate E 2 lysine residue (3). Lipoate attachment is also required for activity of the glycine cleavage enzyme (GCV, the plant enzyme is often called glycine decarboxylase) (4, 5) and the branched chain ␣-keto acid dehydrogenase complex (2).All lipoate attachment was thought to proceed by the classical ATP-dependent mechanism, and Escherichia coli contains such a lipoate ligase (LplA protein) that functions primarily in utilization of exogenously supplied lipoic acid (6, 7). E. coli strains having null mutations in the lplA gene lack the ability to attach exogenously added lipoic acid to protein and also lack detectable ATP-dependent lipoate ligase activity (6, 8). However, these strains retain the ability to attach...

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“…13,17). Initial studies of the regulatory properties of plant mPDC have been conducted in vitro using preparations that had been purified 20-to 300-fold (18)(19)(20)(21). Recently, we (2)(3)(4)16) have developed a method for assaying mPDC activity in situ using purified intact pea seedling mitochondria.…”

mentioning

confidence: 99%

“…2Abbreviations: mPDC, mitochondrial pyruvate dehydrogenase complex; OAA, oxalacetic acid; PDC, pyruvate dehydrogenase complex; INT, 2(p-iodophenyl)-3-p-nitrophenyl-5-phenyl-tetrazolium chloride; Cof, cofactors. are quite sensitive in vitro to product inhibition (17,18,24). At a constant total CoA concentration, a linear decrease in CoA-SH/acetyl-CoA results in a linear decrease in PDC activity.…”

mentioning

confidence: 99%

Acetyl-Coenzyme A Can Regulate Activity of the Mitochondrial Pyruvate Dehydrogenase Complex in Situ

Budde

Fang²,

Randall³

et al. 1991

Plant Physiol.

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In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon Km and K1 relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [19871 Plant Physiol 83: 306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or Lcamitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.The mPDC' occupies a crucial branch point in intermediary metabolism, and activity is regulated by several mechanisms including compartmentation, product and metabolite inhibition, and reversible phosphorylation (reviewed in refs. 13,17). Initial studies of the regulatory properties of plant mPDC have been conducted in vitro using preparations that had been purified 20-to 300-fold (18-21). Recently, we (2-4, 16) have developed a method for assaying mPDC activity in situ using purified intact pea seedling mitochondria. Using this method, several observations on the regulation by reversible phosphorylation have been clarified and extended (2-4). This report continues our investigation of the role of product inhibition in regulation of PDC activity within intact mitochondria.Much like mammalian and microbial PDCs, plant mPDCs

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Plant pyruvate dehydrogenase complex purification, characterization and regulation by metabolites and phosphorylation

Cited by 58 publications

References 32 publications

Lipoic Acid-Dependent Oxidative Catabolism of α-Keto Acids in Mitochondria Provides Evidence for Branched-Chain Amino Acid Catabolism in Arabidopsis

Lipoic Acid-Dependent Oxidative Catabolism of α-Keto Acids in Mitochondria Provides Evidence for Branched-Chain Amino Acid Catabolism in Arabidopsis

A New Metabolic Link

Acetyl-Coenzyme A Can Regulate Activity of the Mitochondrial Pyruvate Dehydrogenase Complex in Situ

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