Subunit binding in the pyruvate dehydrogenase complex from bovine kidney and heart

Wu, Tyzz Liang; Reed, Lester J.

doi:10.1021/bi00297a008

Cited by 31 publications

(8 citation statements)

References 24 publications

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“…The earlier studies demonstrated a greater affinity of the active E1 form to the E2 sites compared with the phosphorylated form (38,46). Our results are in support of these findings and suggest that amino acid substituted mutants of site 1 can not easily replace the wild-type E1 when interacting with E2 (Fig.…”

Section: Discussionsupporting

confidence: 90%

Probing the Mechanism of Inactivation of Human Pyruvate Dehydrogenase by Phosphorylation of Three Sites

Korotchkina

Patel

2001

Journal of Biological Chemistry

132

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Activity of the mammalian pyruvate dehydrogenase complex (PDC) is regulated by phosphorylation-dephosphorylation of three serine residues (designated site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) in the ␣ subunit of the pyruvate dehydrogenase (E1) component. Substitutions of the phosphorylation sites were generated by site-directed mutagenesis. Glutamate (S1E) and aspartate (S1D) substitutions at site 1 resulted in the complete loss of PDC activity; however, these mutants were variably active in the decarboxylation and 2,6-dichlorophenolindophenol assays. S1Q had only 3% of wild-type PDC activity. The apparent K m values for pyruvate increased for the mutants of site 1 when determined in the 2,6-dichlorophenolindophenol assay. The substitutions at sites 2 and 3 caused only moderate reductions in activity in the three assays. S3E had a 27-fold increase in the apparent K m for thiamine pyrophosphate and 8-fold increase in the K i for pyrophosphate. Site 3 was almost completely protected from phosphorylation by thiamine pyrophosphate. The results show that the size rather than negative charge of the substituted amino acid residue affects the active site of E1 and that modification of each of the three serine residues affect the active site in a site-specific manner for its ability to bind the cofactor and substrates.The mammalian pyruvate dehydrogenase complex (PDC) 1 plays an important role in defining the fate of three-carbon compounds derived largely from carbohydrates and to some extent from amino acids. PDC comprises multiple copies of three catalytic enzymes, namely pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3); in addition, the complex also contains a binding protein referred to as E3-binding protein (E3BP), and two regulatory enzymes, namely pyruvate dehydrogenase kinase (PDK) and phosphopyruvate dehydrogenase phosphatase. E1 carries out the decarboxylation of pyruvic acid and the reductive acetylation of lipoyl moieties covalently linked to E2. E2 then catalyzes the transfer of acetyl groups to CoA, forming acetyl-CoA and fully reduced lipoyl moieties. E3 reoxidizes the reduced lipoyl groups of E2 and transfers the electrons to NAD ϩ , forming NADH. Sixty subunits of mammalian E2 and 12 subunits of E3BP form the inner core structure, whereas 20 -30 tetrameric E1 (␣ 2 ␤ 2 ) components bind this core and 6 E3 homodimers bind to the E3BP subunits (1, 2).Regulation of mammalian PDC activity is accomplished in large part by phosphorylation (resulting in inactivation) of the E1 component by a family of pyruvate dehydrogenase kinases (PDK 1-4 isozymes) and dephosphorylation (leading to activation) of phosphorylated E1 by a set of specific phosphatases (phosphopyruvate dehydrogenase phosphatase 1-2 isozymes) (1, 3-6). The ␣ subunit of the E1 component has three phosphorylation sites, named site 1 (Ser-264), site 2 (Ser-271), and site 3 (Ser-203), and phosphorylation of any one of these three sites results in inactivation (7-9). In vivo inactivati...

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Section: Discussionsupporting

confidence: 90%

Probing the Mechanism of Inactivation of Human Pyruvate Dehydrogenase by Phosphorylation of Three Sites

Korotchkina

Patel

2001

Journal of Biological Chemistry

132

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“…A greater affinity of the active non-phosphorylated form of the dehydrogenase (El) component for the E2 core has also been reported for pyruvate dehydrogenase complex (Pratt et al, 1979;Wu & Reed, 1984). The results here indicate this may also be true of branched-chain 2-oxo acid dehydrogenase complex.…”

Section: Discussionsupporting

confidence: 72%

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex

Cook¹,

Bradford²,

Yeaman³

1985

Biochemical Journal

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Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E 1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaCl. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of El and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active El has an approx. 6-fold higher affinity for E2 than does phosphorylated El. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.Branched-chain 2-oxo acid dehydrogenase complex is a multienzyme complex catalysing the committed step in the breakdown of the essential amino acids leucine, isoleucine and valine. This complex is analogous to the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, and comprises an acyltransferase core (E2) around which are arranged the 2-oxo acid dehydrogenase (El) and dihydrolipoamide dehydrogenase (E3) components. In each complex the enzymes act in sequence to complete the oxidative decarboxylation of their respective substrates. As with the pyruvate dehydrogenase complex, the E I component of the branched-chain 2-oxo acid dehydrogenase complex consists of two polypeptides, termed a and fi. The activity of the complex can be controlled by phosphorylation/dephosphorylation of the a-subunit of the El component by a kinase intrinsic to the complex (Fatania et al., 1981;Odessey, 1982;Lawson et al., 1983). The kinase phosphorylates two sites closely grouped on the asubunit, and the amino acid sequence surrounding these sites has been determined, inactivation correlating closely with phosphorylation of one site (Cook et al., 1984). The amino acid sequence surrounding this site shows a distinct homology with Abbreviation used: SDS, sodium dodecyl sulphate.

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“…From D20,w = RT(S20,wAfr(l -vp20,w) and eq 3, an rH = 23.0 nm is calculated for both complexes. Our best estimate of M, of our kidney preparations is 7.5 X 106 based on the assumption that there are 60 E2 subunits and of studies to estimate the level of each of the other components: E1, Cate et al (1979); E3, Wu and Reed (1984); and kinase, Rahmatuallah et al (1987); protein X, Jilka et al (1986). Our preparations of kidney complex give an S°20vl of 73.…”

mentioning

confidence: 85%

Sizing of bovine heart and kidney pyruvate dehydrogenase complex and dihydrolipoyl transacetylase core by quasielastic light scattering

Roche¹,

Powers‐Greenwood²,

Shi³

et al. 1993

Biochemistry

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Quasielastic light scattering (QELS) measurements on several preparations of bovine heart and kidney pyruvate dehydrogenase complex yielded hydrodynamic radii (rH values) ranging from 25.7 to 30 nm. Gel filtration chromatography removed stable aggregates and generated preparations that gave essentially the same rH values of 24.3 +/- 0.6 nm for both complexes. The data were characteristic of a monodisperse system and agree with estimates using cryoelectron microscopy [Wagenknecht et al. (1991) J. Biol. Chem. 266, 24650-24656]. The equivalent hydrodynamic sizes for the heart and kidney complex indicate that the larger number of pyruvate dehydrogenase components in the heart complex (M(r) congruent to 9 x 10(6)) than the kidney complex (M(r) congruent to 7.5 x 10(6)) associate without radial expansion of the heart complex. That accommodation of additional mass is consistent with the space available since even in the more massive complex greater than 80% of the volume within the dimensions of the complex must be occupied by solvent. Preparations of the core of the complex are primarily composed of 60 dihydrolipoyl acetyltransferase (E2) subunits whose inner domains associate to form a pentagonal dodecahedron that is readily observed by electron microscopy (particle radius 10.7-11.3 nm). However, the bulk of E2's mass is present in an exterior multidomain structure. These mobile outer structures are very difficult to observe by standard electron microscopy techniques. Preparations of the core formed stable aggregates that were removed by gel filtration chromatography. QELS measurements gave an rH of 20.1 +/- 0.8 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

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Subunit binding in the pyruvate dehydrogenase complex from bovine kidney and heart

Cited by 31 publications

References 24 publications

Probing the Mechanism of Inactivation of Human Pyruvate Dehydrogenase by Phosphorylation of Three Sites

Probing the Mechanism of Inactivation of Human Pyruvate Dehydrogenase by Phosphorylation of Three Sites

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex

Sizing of bovine heart and kidney pyruvate dehydrogenase complex and dihydrolipoyl transacetylase core by quasielastic light scattering

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