Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase

Tovar‐Méndez, Alejandro; Hirani, Tripty A.; Miernyk, Ján A.; Randall, Douglas D.

doi:10.1016/j.abb.2004.10.017

Cited by 12 publications

(12 citation statements)

References 36 publications

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“…Recombinant His6-AtPDK was prepared as described previously (Tovar-Méndez et al, 2005). The synthetic peptide-based KiC assay (Huang et al, 2010) was used with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

Ahsan¹,

Swatek²,

Zhang³

et al. 2012

Front. Plant Sci.

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The mitochondrial pyruvate dehydrogenase complex (mtPDC) is regulated by reversible seryl-phosphorylation of the E1α subunit by a dedicated, intrinsic kinase. The phospho-complex is reactivated when dephosphorylated by an intrinsic PP2C-type protein phosphatase. Both the position of the phosphorylated Ser-residue and the sequences of the flanking amino acids are highly conserved. We have used the synthetic peptide-based kinase client (KiC) assay plus recombinant pyruvate dehydrogenase E1α and E1α-kinase to perform “scanning mutagenesis” of the residues flanking the site of phosphorylation. Consistent with the results from “phylogenetic analysis” of the flanking sequences, the direct peptide-based kinase assays tolerated very few changes. Even conservative changes such as Leu, Ile, or Val for Met, or Glu for Asp, gave very marked reductions in phosphorylation. Overall the results indicate that regulation of the mtPDC by reversible phosphorylation is an extreme example of multiple, interdependent instances of co-evolution.

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“…Recombinant His6-AtPDK was prepared as described previously (Tovar-Méndez et al, 2005). The synthetic peptide-based KiC assay (Huang et al, 2010) was used with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

Ahsan¹,

Swatek²,

Zhang³

et al. 2012

Front. Plant Sci.

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“…Individual transformants were screened for high-level expression of soluble AtPDC E1. Cell growth, lysis, and affinity purification of recombinant AtPDC E1 and AtPDK are described elsewhere [30]. …”

Section: Methodsmentioning

confidence: 99%

“…Primers DDR471 and DDR472 were used to amplify WT AtPDK [30], while primers DDR473 and DDR474 were used to amplify both WT and mutant forms of E1 α . The AtPDK sequence was cloned into pBT, and the AtE1 α constructs were cloned into pTRG.…”

Section: Methodsmentioning

confidence: 99%

Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

et al. 2011

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We have developed an in vitro system for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinant Arabidopsis thalianaα2β2-heterotetrameric pyruvate dehydrogenase (E1) plus A. thaliana E1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes in kcat, Km, and kcat/Km values. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α.

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“…To calibrate the ELISA, recombinant heterotetrameric A. thaliana E1 was prepared as previously described (Szurmak et al 2003, Tovar‐Mendez et al 2005). For phosphorylation of E1, 1.38 mg of the recombinant protein was incubated overnight at room temperature with 0.044 mg of recombinant AtPDK plus 3 m M ATP.…”

Section: Methodsmentioning

confidence: 99%

“…The PDK is unusual, having a sequence homologous with protein His‐kinases, but phosphorylating only Ser residues in the E1α‐subunit of PDH (Steussy et al 2001, Thelen et al 2000). Biochemical analyses verified that the PDK does not use a P‐His reaction intermediate (Tovar‐Mendez et al 2002), and recently a catalytic mechanism has been proposed (Tovar‐Mendez et al 2005). The PDK is associated with the PDC by specifically binding to the inner lipoyl‐bearing domain (L2) of the dihydrolipoyl acetyltransferase (E2) core (Tuganova et al 2002).…”

Section: Introductionmentioning

confidence: 97%

Is there a signal transduction pathway that links events at the plasma membrane to the phosphorylation state of the mitochondrial pyruvate dehydrogenase complex?

Miernyk

Szurmak

Tovar‐Méndez

et al. 2006

Physiologia Plantarum

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Monoclonal antibodies against the E1a subunit of pyruvate dehydrogenase (PDH) were used to quantify the mitochondrial pyruvate dehydrogenase complex (mtPDC) by enzyme-linked immunosorbent assay (ELISA). Recombinant Arabidopsis thaliana PDH (E1) was used to calibrate the ELISA. Antibodies against a synthetic phosphopeptide corresponding to phosphorylation site one of E1a were used in an ELISA to quantify phospho-PDC (P-PDC). For calibration of the second ELISA, recombinant E1 was phosphorylated in vitro with recombinant A. thaliana E1-kinase. The two ELISA were used to quantify mitochondrial total-and P-PDC in clarified homogenates from Nicotiana tabacum BY-2 suspension cells. The level of mtPDC remained constant throughout the 7-day growth cycle at 25.1 g 21 FW. During the lag (days 0-2) and stationary (day 7) stages of the growth cycle, the mtPDC was completely phosphorylated (inactive), whereas during the log-growth stage it was completely dephosphorylated (active). Exposure of 3-or 7-day posttransfer suspension cells to osmotic stress significantly decreased proportion of P-PDC. A series of pharmacological studies were undertaken to gain insight into the signal transduction pathways coupling osmotic stress perception with control of mitochondrial respiration. Results from these studies indicate a signal transduction pathway linking stress perception to control of mitochondrial respiration that includes protein kinases and phosphoprotein phosphatases.Abbreviations -E1 or PDH, pyruvate dehydrogenase; E2, dihydrolipoyl acetyltransferase; ELISA, enzyme-linked immunosorbent assay; L2, the E2 inner lipoyl domain; mt, mitochondrial; P-, phospho; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase.

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Analysis of the catalytic mechanism of pyruvate dehydrogenase kinase

Cited by 12 publications

References 36 publications

“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

“Scanning mutagenesis” of the amino acid sequences flanking phosphorylation site 1 of the mitochondrial pyruvate dehydrogenase complex

Asp295 Stabilizes the Active-Site Loop Structure of Pyruvate Dehydrogenase, Facilitating Phosphorylation of Ser292 by Pyruvate Dehydrogenase-Kinase

Is there a signal transduction pathway that links events at the plasma membrane to the phosphorylation state of the mitochondrial pyruvate dehydrogenase complex?

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