Plant pyruvate dehydrogenase complex: Analysis of the kinetic properties and metabolite regulation

1987

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ABSTRACIThe pyruvate dehydrogenase complex was isolated, partially purified, and characterized from green pea (Pisum sativum L., cv Little Marvel) leaf mitochondria. The pH optimum for the overall reaction was 7.6. The divalent cation requirement was best satisfied by Mg2". Reaction velocity was maximal at 40°C. Pyruvate was a better substrate than 2-oxobutyrate; other 2-oxo-acids were not substrates. Michaelis constants for substrates were; pyruvate, 57 micromolar, NAD, 122 micromolar, Coenzyme-A, 5 micromolar, Mg2", 0.36 millimolar, Mg-thiamine pyrophosphate, 80 nanomolar. The products, NADH and acetyl-Coenzyme-A, were linear competitive inhibitors with respect to NAD and Coenzyme A. Inhibition constants were 18 and 10 micromolar, respectively. Glyoxylate inhibited complex activity only in the absence of thiol reagents. Glyoxylate inhibition was competitive with respect to pyruvate with an inhibition constant of 51 micromolar. Among mitochondrial metabolites examined as potential effectors, only ADP with an inhibition constant of 0.57 millimolar could be of physiological significance.

Section: Resultssupporting

confidence: 76%

Some Kinetic and Regulatory Properties of the Pea Mitochondrial Pyruvate Dehydrogenase Complex

Miernyk¹,

1987

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“…The NH4' produced by photorespiration is considered to be assimilated through the glutamine synthase/glutamate synthase cycle and GDH is thought not to be involved (1 1,14,21,23 (12,20). Purified, intact mitochondria cannot oxidize pyruvate without added TPP (3).…”

Section: Mm)mentioning

confidence: 99%

Regulation of Pea Mitochondrial Pyruvate Dehydrogenase Complex

Schuller¹,

1989

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Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentraffons of Na+. NH4+, at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K+ in the range I to 10 millimolar also enhanced inactivation. In contrast, Na+ was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NHW+ is discussed in relation to a possible regulatory interaction between photorespiratory NH.+ production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.inhibited by TPP in dialyzed extracts but not in intact mitochondria. Furthermore, K+ and NH4+ are shown to markedly stimulate PDH kinase activity with NH4+ being more effective than K+. The latter result is discussed in relation to a potential role for photorespiratory NHE+ in regulating PDC activity and consequently the carbon supply for dark respiration during photosynthesis. This is important because it is not known to what extent dark respiration proceeds simultaneously with photosynthesis, let alone what regulates the balance between these two processes in meeting energy demands (9). MATERIALS AND METHODS BiochemicalsBiochemicals were purchased from either Sigma (St. Louis, MO) or Pharmacia (Piscataway, NJ). ATP and pyruvate were sodium salts and TPP was the free acid.The mitochondrial pyruvate dehydrogenase complex (PDC2) catalyzes a key reaction regulating entry of carbon into the tricarboxylic acid cycle (4) and thereby indirectly affects the rate of dark respiration. Activity of the complex is regulated in part by reversible phosphorylation and in part by product inhibition (4,12,17,19

“…Reid et al (18) also observed only a minor stimulation of Ricinus endosperm plastid PDC with TPP. On the other hand, Rubin et al (20) proposed possible regulation of mitochondrial PDC by TPP because of its very rapid dissociation. Regulation of this type seems improbable for the plastid complex.…”

Section: Fraction Number Figmentioning

confidence: 99%

“…Mitochondrial PDCs from plant sources (19,20) require the addition of TPP for maximal activity, indicating the ready dissociation ofTPP from the enzyme complex. In plastids, however, a tighter association appears to exist.…”

Section: Fraction Number Figmentioning

confidence: 99%

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Purification and Characterization of the Pea Chloroplast Pyruvate Dehydrogenase Complex

Camp

1985

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140

The pyruvate dehydrogenase complex has been purified 76-fold, to a specific activity of0.6 umoles per minute per milligram protein, beginning with isolated pea (Pisum sativum L. var Little Marvel) chloroplasts. Purification was accomplished by rate zonal sedimentation, polyethyleneglycol precipitation, and ethyl-agarose affinity chromatography. Characterization of the substrates as pyruvate, NAD', and coenzyme-A and the products as NADH, C02, and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruvate dehydrogenase complex. Immunochemical analysis demonstrated significant differences in structure and organization between the chloroplast pyruvate dehydrogenase complex and the more thoroughly characterized mitochondrial complex. Chloroplast complex has a higher magnesium requirement and a more alkaline pH optimum than mitochondrial complex, and these properties are consistent with light-mediated regulation in vivo. The chloroplast pyruvate dehydrogenase complex is not, however, regulated by ATP-dependent inactivation. The properties and subcellular localization of the chloroplast pyruvate dehydrogenase complex are consistent with its role of providing acetyl-CoA and NADH for fatty acid synthesis.The pyruvate dehydrogenase complex is a multicomponent system composed ofthe three enzymes, pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. The (11,15,16). Phosphorylation (inactivation) of pyruvate dehydrogenase is catalyzed by a tightly associated kinase and dephosphorylation (activation) by a more loosely associated phosphatase (10).The pivotal position of PDC in metabolism has resulted in a vast amount of research on the enzyme from nonplant tissues. By comparison, relatively little is known about plant PDC, and most information that is available concerns the mitochondrial form of the enzyme. The limited knowledge of plastid PDC prompted the current investigation with the purpose ofestablishing the chloroplast enzyme as a true PDC with properties distinct from the mitochondrial enzyme and consistent with its postulated role in chloroplast fatty acid biosynthesis.