Isolation and Characterisation of Pyruvate Dehydrogenase Complex from Brewer’s Yeast

Wais, U; Gillmann, Ursula; Ullrich, Johannes

doi:10.1515/bchm2.1973.354.2.1378

Cited by 26 publications

(9 citation statements)

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“…In the absence of alternative oxidase (CON), the activity of ICDH decreased as the specific growth rate approached D crit , an effect likely due to inhibition caused by an increasing NADH/NAD ratio, as observed previously for Yarrowia lipolitica (30). Allosteric inhibition of pyruvate dehydrogenase (31,32) and of other key TCA cycle enzymes (ICDH, ␣-ketoglutarate dehydrogenase, and malate dehydrogenase) by NADH (or by the NADH/NAD ratio) restricts the entry of glycolytic carbon from pyruvate into the mitochondria. Under these circumstances, carbon is shunted to acetaldehyde, and subsequently to ethanol, by coordinated action of pyruvate decarboxylase and NAD-dependent aldehyde dehydrogenases, routes that avoid additional NADH accumulation.…”

Section: Discussionsupporting

confidence: 62%

Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

Vemuri

Eiteman

McEwen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

321

273

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Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as ''overflow metabolism'' or ''the Crabtree effect.'' The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.alternative oxidase ͉ Crabtree effect ͉ NADH oxidase ͉ redox metabolism R edox homeostasis is a fundamental requirement for sustained metabolism and growth in all biological systems. The intracellular redox potential is primarily determined by the NADH/NAD ratio and to a lesser extent by the NADPH/NADP ratio. In Saccharomyces cerevisiae, Ͼ200 reactions involve these cofactors spread over a large spectrum of cellular functions (1). Because NADH is a highly connected metabolite in the metabolic network (1), any change in the NADH/NAD ratio leads to widespread changes in metabolism (2). NADH is generated primarily in the cytosol by glycolysis and in the mitochondria by the tricarboxylic acid (TCA) cycle. Because the NADH/NAD redox couple cannot traverse the mitochondrial membrane in S. cerevisiae and other eukaryotic cells (3), distinct mechanisms oxidize NADH to NAD in the cytosol and mitochondria. Cytosolic NADH is oxidized by two external (cytosolic) mitochondrial membrane-bound NADH dehydrogenases encoded by NDE1 and NDE2 genes with catalytic sites facing the cytosol (4). Additionally, glycerol-3-phosphate dehydrogenases (encoded by GPD1 and GPD2) oxidize cytosolic NADH with concomitant glycerol formation when the NADH formation rate surpasses its oxidation rate (5). Mitochondrial NADH is oxidized by one internal mitochondrial membrane-bound NADH dehydrogena...

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Section: Discussionsupporting

confidence: 62%

Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

Vemuri

Eiteman

McEwen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

321

273

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“…Both are absolutely specific for NAD+ as the electron acceptor, there being no activity with NADP+. This is the same as the complex from other sources which are also specific for NAD+ (23). The mitochondrial complex is also absolutely specific for pyruvate as the keto acid, and shows no activity with a range of keto acids including a-ketoglutarate, oxaloacetate, and glyoxylate.…”

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confidence: 53%

“…The pH optimum of the mammalian complex is between pH 8 and 9 (9). However, that of the brewers' yeast complex (23) and the Neurospora complex (7) There is an absolute requirement for TPP in the case of the partially purified mitochondrial complex, and TPP also increases the activity of the proplastid complex, indicating that TPP can dissociate. At higher concentrations, TPP inhibits the proplastid complex (Fig.…”

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confidence: 99%

Pyruvate Dehydrogenase Complex from Higher Plant Mitochondria and Proplastids

Reid¹,

Thompson²,

Lyttle³

et al. 1977

Plant Physiol.

112

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The pyruvate dehydrogenase complex from pea (Pisum sativum L.) mitochondria was purified 23-fold by high speed centrifugation and glycerol gradient fractionation. The complex had a s20,,0, of 47.5S but this is a minimal value since the complex is unstable. The complex is specific for NAD+ and pyruvate; NADP+ and other keto acids give no reaction.Mg2+, thiamine pyrophosphate, and cysteine are also required for maximal activity. The pH optimum for the complex was between 6.5 and 7.5.Continuous sucrose density gradients were used to separate castor bean (Ricinus communis L.) endosperm proplastids from mitochondria.Pyruvate dehydrogenase complex activity was found to be coincident with the proplastid peak on all of the gradients. Some separation of proplastids and mitochondria could be achieved by differential centrifugation and the ratios of the activities of the pyruvate dehydrogenase complex to succinic dehydrogenase and acetyl-CoA carboxylase to succinic dehydrogenase were consistent with both the pyruvate dehydrogenase complex and acetyl-CoA carboxylase being present in the proplastid. The proplastid fraction has to be treated with a detergent, Triton X-100, before maximal activity of the pyruvate dehydrogenase complex activity is expressed, indicating that it is bound in the organelle. The complex had a sharp pH optimum of 7.5. The complex required added Mg2+, cysteine, and thiamine pyrophosphate for maximal activity but thiamine pyrophosphate was inhibitory at higher concentrations. The sequence of reactions catalyzed by these enzymes is:

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“…This enzyme may not obey normal kinetics under all substrate conditions (2). NADH appears to be competitive with NAD+ in most systems (5,8,17).…”

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confidence: 99%

Pyruvate Dehydrogenase Complex from Higher Plant Mitochondria and Proplastids: Kinetics

Thompson¹,

Reid²,

Lyttle³

et al. 1977

Plant Physiol.

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be competitive with the substrate for that enzyme unless the central complexes are kinetically important, in which case they will be noncompetitive. This prediction has been confirmed for acetyl-CoA and NADH in the bovine kidney and pig heart complexes (7, 16). Other product inhibition patterns should be uncompetitive except for the interaction of one product and the substrate of the subsequent enzyme when it will be noncompetitive, if the reaction at that site is random sequential. In the mammalian complexes (7, 16), acetyl-CoA and NADH are both uncompetitive against pyruvate but acetyl-CoA and NADH are noncompetitive against NAD+ and CoA, respectively. This may indicate that these products can act as dead end inhibitors, or the binding of products may sterically hinder the binding of substrates on the adjacent enzyme. A third possibility is that there may be protein-protein interactions between the second and third enzymes of the complex (7, 16).The steady-state kinetic analyses to determine the reaction mechanism of the plant complex have not been described. The regulatory properties of the enzyme from potato tubers have been described and some preliminary kinetics performed (5). Since some of the interactions between products and substrates are other than those predicted by theory, it is important that further kinetics be performed. Also, a steady-state kinetic analysis may be used to determine whether the mitochondrial enzyme and the enzyme from proplastids (11) are significantly different.The pyruvate dehydrogenase complex consists of three enzymes: pyruvate dehydrogenase (pyruvate lipoate oxidoreductase, EC 1.2.4.1), lipoate transacetylase (acetyl-CoA dihydrolipoate S-acetyl transferase, EC 2.3.1.12), and dihydrolipoamide dehydrogenase (NADH lipoamide oxidoreductase, EC 1.6.4.3).The reactions of these three enzymes are shown in the accompanying paper (11) which also gives an account of the purification and preliminary characteristics of the pea mitochondrial complex and isolation of the proplastid complex. The reactions of the complex occur at three distinct sites on the complex located on three different proteins. The nature of the reactions at each of these sites suggests that the over-all mechanism should be ping pong. A rate equation for a three-site ping-pong mechanism has been derived by Cleland (4) and from this it was predicted that the pyruvate dehydrogenase complex should show initial substrate velocity patterns which are parallel when plotted in double reciprocal form, regardless of which substrate is varied at fixed levels of a second. Such a pattern has been demonstrated for the pyruvate dehydrogenase complexes from bovine kidney (16) and pig heart (7). The products of each enzyme component should I

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Isolation and Characterisation of Pyruvate Dehydrogenase Complex from Brewer’s Yeast

Cited by 26 publications

References 0 publications

Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

Pyruvate Dehydrogenase Complex from Higher Plant Mitochondria and Proplastids

Pyruvate Dehydrogenase Complex from Higher Plant Mitochondria and Proplastids: Kinetics

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