Some Properties of Pea Mitochondrial Phospho-Pyruvate Dehydrogenase-Phosphatase

Miernyk, Ján A.; Randall, Douglas D.

doi:10.1104/pp.83.2.311

Cited by 45 publications

(29 citation statements)

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“…Although the kinetics of inactivation were slower than observed in previous investigations of plant mitochondrial PDCs [5,6,21], recombinant AtPDK was capable of inactivating 90 % of kinasedepleted maize mitochondrial PDC. The specific activity of MBP-AtPDK was lower (10 PDC units inactivated\min per mg of PDK) than that of maize MBP-PDK2 (40 PDC units inactivated\min per mg of PDK [18]) with maize mitochondrial PDC as substrate.…”

Section: Atpdk Inactivates Kinase-depleted Maize Mitochondrial Pdc Ancontrasting

confidence: 38%

“…PDK curtails mitochondrial PDC activity by catalysing the transfer of the γ-phosphate from ATP to the α subunit of the pyruvate dehydrogenase (E1) component enzyme [4,5]. Conversely, phospho-pyruvate dehydrogenase phosphatase reactivates this component by catalysing dephosphorylation in a Mg# + -dependent manner [6]. The phosphorylation state, and hence the activity status, of PDC is determined by the net sum of these opposing enzyme activities.…”

Section: Introductionmentioning

confidence: 99%

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Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

Thelen

Miernyk

Randall

2000

Biochemical Journal

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Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.

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Section: Atpdk Inactivates Kinase-depleted Maize Mitochondrial Pdc Ancontrasting

confidence: 38%

Section: Introductionmentioning

confidence: 99%

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

Thelen

Miernyk

Randall

2000

Biochemical Journal

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“…The first plant enzyme shown to be regulated by reversible phosphorylation was the mitochondrial pyruvate dehydrogenase complex [PDC (15,16)]. Associated with the complex are the regulatory converter enzymes (pyruvate dehydrogenase kinase and phosphatase) which phosphorylate (inactivate) and dephosphorylate (activate) PDC (13,17). The pyruvate dehydrogenase protein phosphatase associated with the complex requires millimolar concentrations of Mg2" and the Mg2"-dependent activation is inhibited by Ca2" (2).…”

Section: Discussionmentioning

confidence: 99%

Inactivation of Highly Activated Spinach Leaf Sucrose-Phosphate Synthase by Dephosphorylation

Huber

Hite²,

Outlaw³

1991

Plant Physiol.

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Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be phosphorylated and inactivated in vitro with ['-32P]ATP Arch Biochem Biophys 270: 681-690). Thus, it was surprising to find that SPS, extracted from leaves fed mannose in the light to highly activate the enzyme, could be inactivated in an ATP-independent manner when desalted crude extracts were preincubated at 250C before assay. The "spontaneous" inactivation involved a loss in activity measured with limiting substrate concentrations in the presence of the inhibitor, Pi, without affecting maximum catalytic activity. The spontaneous inactivation was unaffected by exogenous carrier proteins and protease inhibitors, but was inhibited by inorganic phosphate, fluoride, and molybdate, suggesting that a phosphatase may be involved. Okadaic acid, a potent inhibitor of mammalian type 1 and 2A protein phosphatases, had no effect up to 5 micromolar. Inactivation was stimulated about twofold by exogenous Mg2+ and was relatively insensitive to Ca2+ and to pH over the range pH 6.5 to 8.5. Radioactive phosphate incorporated into SPS during labeling of excised leaves with [32PJPi (initially in the dark and then in the light with mannose) was lost with time when desalted crude extracts were incubated at 250C, and the loss in radiolabel was substantially reduced by fluoride. These results provide direct evidence for action of an endogenous phosphatase(s) using SPS as substrate. We postulate that highly activated SPS contains phosphorylated residue(s) that increase activation state, and that spontaneous inactivation occurs by removal of these phosphate group(s). Inactivation of SPS in vivo caused by feeding uncouplers to darkened leaf tissue that had previously been fed mannose in the dark, may occur by this mechanism. However, there is no evidence that this mechanism is involved in light-dark regulation of SPS in vivo.Regulation of SPS2 activity is thought to be one of the components that controls the flux of carbon into sucrose, and hence translocation, in situ (for review see ref. 19 (9). Thus, phosphorylation of SPS occurred both in vivo and in vitro, and in both systems, the more highly phosphorylated enzyme had a lower activation state. Attempts to study the biochemical mechanism involved have been successful because the spinach leaf enzyme is often relatively stable (for short periods of time) at room temperature. The work we report here focused on the highly activated enzyme extracted from illuminated leaves that were fed mannose. We compared this SPS form with the relatively inactive (extracted from darkened leaves) and intermediate enzymeforms (extracted from leaves illuminated without mannose). We observed that highly activated enzyme was not stable when desalted crude extracts were preincubated at 25°C. Our study of this loss in activation state indicates that the "spontaneous" inactivation is mediated by an endogenous phosphatase(s), which constitutes evidence for a novel regulatory covalent modification of SPS.

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“…Associated with the mitochondrial complex are two regulatory enzymes (PDH kinase and PDH-P phosphatase) which catalyze the reversible phosphorylation of the E,-a subunit of PDH (7,9,(17)(18)(19)(20). Phosphorylation results in inactivation and dephosphorylation reactivates PDC (9,13). Chloroplastic PDC is not regulated by phosphorylation (3,25).…”

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confidence: 99%

“…Most in vitro investigations of PDC regulation have utilized partially purified PDC (13). Those studies would suggest that the ' This research was supported in part by the Missouri Agriculture Experimental Station, National Science Foundation grant DMB-8506473, and the Food for the 21st Century Program.…”

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confidence: 99%

Regulation of Steady State Pyruvate Dehydrogenase Complex Activity in Plant Mitochondria

Budde

Randall²

1988

Plant Physiol.

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The requirements for reactivation (dephosphorylation) of the pea (Pisum sativum L.) leaf mitochondrial pyruvate dehydrogenase complex (PDC) were studied in terms of magnesium and ATP effects with intact and permeabilized mitochondria. The requirement for high concentrations of magnesium for reactivation previously reported with partially purified PDC is shown to affect inactivation rather than reactivation. The observed rate of inactivation catalyzed by pyruvate dehydrogenase (PDH) kinase is always greater than the reactivation rate catalyzed by PDH-P phosphatase. Thus, reactivation would only occur if ATP becomes limiting. However, pyruvate which is a potent inhibitor of inactivation in the presence of thiamine pyrophosphate, results in increased PDC activity. Analysis of the dynamics of the phosphorylation-dephosphorylation cycle indicated that the covalent modification was under steady state control. The steady state activity of PDC was increased by addition of pyruvate. PDH kinase activity increased threefold during storage of mitochondria suggesting that there may be an unknown level of regulation exerted on the enzyme complex.The pyruvate dehydrogenase complex (PDC2) is a large multienzyme complex consisting of three enzymes which convert pyruvate to acetyl-CoA. Associated with the mitochondrial complex are two regulatory enzymes (PDH kinase and PDH-P phosphatase) which catalyze the reversible phosphorylation of the E,-a subunit of PDH (7,9,[17][18][19][20]. Phosphorylation results in inactivation and dephosphorylation reactivates PDC (9, 13). Chloroplastic PDC is not regulated by phosphorylation (3,25).It has been proposed that the phosphorylation-dephosphorylation of the mammalian PDC is regulated in part by ATP:ADP ratios (21, 23) or adenylate energy charge (9) and pyruvate concentration (7, 9). We have reported that phosphorylation of the plant mitochondrial PDC is unaffected by changes in the ATP:ADP ratio (2). We have proposed a mechanism by which the ATP dependent inactivation can be regulated by pyruvate (2); however pyruvate did not affect reactivation.Most in vitro investigations of PDC regulation have utilized partially purified PDC (13). Those (14), and the Km (Mg2") for PDH-P phosphatase is 3.8 mM (13). The ATP concentration in plant leaf mitochondria ranges from 0.4 mm in illuminated tissue to 0.2 mm in nonilluminated green leaf tissue (4, 6) and thus, it would seem that the ATP levels would keep the enzyme phosphorylated. Magnesium concentrations of mammalian mitochondria have been reported to be less than 0.5 mm (12) and if the magnesium concentrations of plant mitochondria are similar to those reported for mammalian mitochondria, it would seem that the dephosphorylation would be insufficient to overcome the action of PDH kinase. Therefore, we have undertaken the study of the dephosphorylation (reactivation) of PDC.In this paper, we have examined the requirement for Mgdependent reactivation and the effect of pyruvate on the steady state PDC activity using functional, intact mitoc...

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Some Properties of Pea Mitochondrial Phospho-Pyruvate Dehydrogenase-Phosphatase

Abstract: Reactivation of the pea mitochondrial pyruvate dehydrogenase complex was the result of dephosphorylation catalyzed by phospho-pyruvate dehydrogenase-phosphatase, an intrinsic component of the complex.

Cited by 45 publications

References 2 publications

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

Inactivation of Highly Activated Spinach Leaf Sucrose-Phosphate Synthase by Dephosphorylation

Regulation of Steady State Pyruvate Dehydrogenase Complex Activity in Plant Mitochondria

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