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Structural and functional insights into the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase kinase 2.

ABSTRACT: PDHK2 is a mitochondrial protein kinase that phosphorylates pyruvate dehydrogenase complex, thereby down-regulating the oxidation of pyruvate. Here, we present the crystal structure of PDHK2 bound to the inner lipoyl-bearing domain of dihydrolipoamide transacetylase (L2) determined with or without bound adenylyl imidodiphosphate. Both structures reveal a PDHK2 dimer complexed with two L2 domains. Comparison with apo-PDHK2 shows that L2 binding causes rearrangements in PDHK2 structure that affect the L2- and E1-binding sites. Significant differences are found between PDHK2 and PDHK3 with respect to the structure of their lipoyllysine-binding cavities, providing the first structural support to a number of studies showing that these isozymes are markedly different with respect to their affinity for the L2 domain. Both structures display a novel type II potassium-binding site located on the PDHK2 interface with the L2 domain. Binding of potassium ion at this site rigidifies the interface and appears to be critical in determining the strength of L2 binding. Evidence is also presented that potassium ions are indispensable for the cross-talk between the nucleotide- and L2-binding sites of PDHK2. The latter is believed to be essential for the movement of PDHK2 along the surface of the transacetylase scaffold.

SUBMITTER: Green T

PROVIDER: S-EPMC2414299 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Structural and functional insights into the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase kinase 2.

Green Todd T Grigorian Alexei A Klyuyeva Alla A Tuganova Alina A Luo Ming M Popov Kirill M KM

The Journal of biological chemistry 20080403 23

PDHK2 is a mitochondrial protein kinase that phosphorylates pyruvate dehydrogenase complex, thereby down-regulating the oxidation of pyruvate. Here, we present the crystal structure of PDHK2 bound to the inner lipoyl-bearing domain of dihydrolipoamide transacetylase (L2) determined with or without bound adenylyl imidodiphosphate. Both structures reveal a PDHK2 dimer complexed with two L2 domains. Comparison with apo-PDHK2 shows that L2 binding causes rearrangements in PDHK2 structure that affect ...[more]

PMID: 18387944

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Project description:1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [(32)P]phosphate from [gamma-(32)P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100mum) and cyclic 3':5'-nucleotides (at 10mum) had no significant effect on kinase activity. 3. The K(m) for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76mum. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The K(m) for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9-25.4mum. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The K(m) for pyruvate in the pyruvate dehydrogenase reaction was 35.5mum. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25-500mum. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10mum) the kinase activity was enhanced by low concentrations of pyruvate (25-100mum) and inhibited by a high concentration (500mum). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms (14)CO(2) from [1-(14)C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg(2+) (15mm) and by Ca(2+) (10nm-10mum) at low Mg(2+) (0.15mm) but not at high Mg(2+) (15mm).

Dataset Information

Structural and functional insights into the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase kinase 2.

Publications

Structural and functional insights into the molecular mechanisms responsible for the regulation of pyruvate dehydrogenase kinase 2.

Similar Datasets

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