Role of the E2 core in the dominant mechanisms of regulatory control of mammalian pyruvate dehydrogenase complex

Roche, Thomas E.; Liu, S.; Ravindran, Sundari; Baker, Jason C.; Wang, L.

doi:10.1007/978-3-0348-8981-0_3

Cited by 12 publications

(31 citation statements)

References 48 publications

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“…A "hand-over-hand" model has been proposed in which both subunits of the PDH kinases interact with different inner lipoyl domains by repeated dissociation and reassociation, thereby allowing the kinase to move along the surface of the 60-meric PDC E2 core (26). It was suggested that this mechanism facilitates the efficient phosphorylation of PDC E1 by the limited number of the kinase molecules on the E2 dodecahedron of PDC (27). In the present study, we show that the BCKD kinase also depends on lipoylated E2 for efficient phosphorylation of BCKD E1, similar to PDH kinases.…”

Section: Discussionmentioning

confidence: 99%

Tetrameric Assembly and Conservation in the ATP-binding Domain of Rat Branched-chain α-Ketoacid Dehydrogenase Kinase

Wynn¹,

Chuang²,

Cote³

et al. 2000

Journal of Biological Chemistry

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We showed previously that the rat branched-chain ␣-ketoacid dehydrogenase (BCKD) kinase is capable of autophosphorylation. However, despite its sequence similarity to bacterial histidine protein kinases, BCKD kinase does not function as a histidine protein kinase. In the present study, we report that the rat BCKD kinase exists as a homotetramer of M r ‫؍‬ 185,000, based on results of gel filtration and dynamic light scattering. This is in contrast to the related mammalian pyruvate dehydrogenase kinase isozymes that occur as homodimers. The tetrameric assembly of BCKD kinase was confirmed by the presence of four 5-adenylyl-imidodiphosphatebinding sites (K D ‫؍‬ 4.1 ؋ 10 ؊6 M) per molecule of the kinase. Incubation of the BCKD kinase with increasing concentrations of urea resulted in dissociation of the tetramer to dimers and eventually to monomers as separated on a sucrose density gradient. Both tetramers and dimers, but not the monomer, maintained the conformation capable of binding ATP and undergoing autophosphorylation. BCKD kinase depends on a fully lipoylated transacylase for maximal activity, but the interaction between the kinase and the transacylase is impeded in the presence of high salt concentrations. Alterations of conserved residues in the ATP-binding domain led to a marked reduction or complete loss in the catalytic efficiency of the BCKD kinase. The results indicate that BCKD kinase, similar to pyruvate dehydrogenase kinase isozymes, belongs to the superfamily of ATPase/kinase.The mammalian mitochondrial branched-chain ␣-ketoacid dehydrogenase (BCKD) 1 complex catalyzes the oxidative decarboxylation of the branched-chain ␣-ketoacid derived from leucine, isoleucine, and valine. The mammalian BCKD complex is a macromolecule (M r ϭ 4 ϫ 10 6 ) comprising multiple copies of five component enzymes. The three catalytic components are as follows: a thiamine pyrophosphate-dependent branchedchain ␣-ketoacid decarboxylase (E1) with two E1␣ (M r ϭ 45, 500) and two E1␤ (M r ϭ 37, 500) subunits; a dihydrolipoyl transacylase (E2), which contains 24 lipoate-bearing polypeptides (monomer, M r ϭ 45,000); and a dihydrolipoyl dehydrogenase (E3) (monomer, M r ϭ 55,000) (1). In addition, the BCKD complex contains two regulatory enzymes, a specific kinase (2) and a specific phosphatase (3), which control enzyme activity through a reversible phosphorylation (inactivation)-dephosphorylation (activation) of E1 (2, 3). The BCKD complex is organized around the 24-mer dihydrolipoyl transacylase cubic core (M r ϭ 1.1 ϫ 10 6 ), to which E1, E3, the kinase, and the phosphatase are attached through ionic interactions.The regulation of the activity of the branched-chain ␣-ketoacid dehydrogenase complex through a kinase-mediated phosphorylation has been extensively studied. Rats fed low protein diets showed low hepatic enzyme complex activity (2). This is associated with a reduction in the activity state, or the percent of total enzyme in the dephosphorylated form, of the enzyme compared with rats fed a normal diet (2, 4). Starvatio...

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Section: Discussionmentioning

confidence: 99%

Tetrameric Assembly and Conservation in the ATP-binding Domain of Rat Branched-chain α-Ketoacid Dehydrogenase Kinase

Wynn¹,

Chuang²,

Cote³

et al. 2000

Journal of Biological Chemistry

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“…Acetyl-CoA and NADH, common products of the PDC reaction and the catabolism of fatty acids, stimulate bovine PDK activity resulting in the feed-back throttling down of PDC activity (2,38,39). Increases in the NADH/NAD ϩ and acetylCoA/CoA ratio stimulate PDK activities by increasing the proportion of reduced and acetylated lipoyl groups on the lipoyl domain of E2 (39 -43).…”

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confidence: 99%

Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase

Baker

Yan

Peng

et al. 2000

Journal of Biological Chemistry

111

194

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Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 M L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k cat along with about a 2-fold decrease in the K m of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.

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“…In the intact multienzyme complexes, E2 noncovalently binds multiple copies of two peripheral enzymes: the thiamin diphosphate-dependent pyruvate decarboxylase and the flavoenzyme dihydrolipoyl dehydrogenase leading to a M r of these systems of up to 10 million Da (1). In eukaryotic organisms, additional proteins may be attached to these multienzyme complexes, among them specific kinases and phosphatases involved in metabolic regulation (3) and dihydrolipoyl dehydrogenase-binding proteins.…”

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confidence: 99%

Principles of quasi-equivalence and Euclidean geometry govern the assembly of cubic and dodecahedral cores of pyruvate dehydrogenase complexes

Izard

Aevarsson

Allen

et al. 1999

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

174

236

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The pyruvate dehydrogenase multienzyme complex (M r of 5-10 million) is assembled around a structural core formed of multiple copies of dihydrolipoyl acetyltransferase (E2p), which exhibits the shape of either a cube or a dodecahedron, depending on the source. The crystal structures of the 60-meric dihydrolipoyl acyltransferase cores of Bacillus stearothermophilus and Enterococcus faecalis pyruvate dehydrogenase complexes were determined and revealed a remarkably hollow dodecahedron with an outer diameter of Ϸ237 Å, 12 large openings of Ϸ52 Å diameter across the fivefold axes, and an inner cavity with a diameter of Ϸ118 Å. Comparison of cubic and dodecahedral

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Role of the E2 core in the dominant mechanisms of regulatory control of mammalian pyruvate dehydrogenase complex

Cited by 12 publications

References 48 publications

Tetrameric Assembly and Conservation in the ATP-binding Domain of Rat Branched-chain α-Ketoacid Dehydrogenase Kinase

Tetrameric Assembly and Conservation in the ATP-binding Domain of Rat Branched-chain α-Ketoacid Dehydrogenase Kinase

Marked Differences between Two Isoforms of Human Pyruvate Dehydrogenase Kinase

Principles of quasi-equivalence and Euclidean geometry govern the assembly of cubic and dodecahedral cores of pyruvate dehydrogenase complexes

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