Recognition of the inner lipoyl-bearing domain of dihydrolipoyl transacetylase and of the blood glucose-lowering compound AZD7545 by pyruvate dehydrogenase kinase 2

Pyruvate dehydrogenase kinase 2 (PDHK2) is a unique mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase multienzyme complex (PDC). PDHK2 is an integral component of PDC tightly bound to the inner lipoyl-bearing domains (L2) of the dihydrolipoyl transacetylase component (E2) of PDC. This association has been reported to bring about an up to 10-fold increase in kinase activity. Despite the central role played by E2 in the maintenance of PDHK2 functionality in the PDC-bound state, the molecular mechanisms responsible for the recognition of L2 by PDHK2 and for the E2-dependent PDHK2 activation are largely unknown. In this study, we used a combination of molecular modeling and site-directed mutagenesis to identify the amino acid residues essential for the interaction between PDHK2 and L2 and for the activation of PDHK2 by E2. On the basis of the results of site-directed mutagenesis, it appears that a number of PDHK2 residues located in its R domain (P22, L23, F28, F31, F44, L45, and L160) and in the so-called "cross arm" structure (K368, R372, and K391) are critical in determining the strength of the interaction between PDHK2 and L2. The residues of L2 essential for recognition by PDHK2 include L140, K173, I176, E179, and to a lesser extent D164, D172, and A174. Importantly, certain PDHK2 residues forming interfaces with L2, i.e., K17, P22, F31, F44, R372, and K391, are also critical for the maintenance of enhanced PDHK2 activity in the E2-bound state. Finally, evidence that the blood glucose-lowering compound AZD7545 disrupts the interactions between PDHK2 and L2 and thereby inhibits PDHK2 activity is presented.     

Diverse mechanisms of inhibition of pyruvate dehydrogenase kinase by structurally distinct inhibitors

The mechanism of action of structurally distinct pyruvate dehydrogenase kinase (PDK) inhibitors was examined in assays with experimental contexts ranging from an intact pyruvate dehydrogenase complex (PDC) with and without supplemental ATP or ADP to a synthetic peptide substrate to PDK autophosphorylation. Some compounds directly inhibited the catalytic activity of PDKs. Some of the inhibitor classes tested inhibited autophosphorylation of recombinant PDK1 and PDK2. During these studies, PDC was shown to be directly inhibited by a novel mechanism; the addition of supplemental recombinant PDKs, an effect that is ADP-dependent and partly alleviated by members of each of the compound classes tested. Overall, these data demonstrate that small molecules acting at diverse sites can inhibit PDK activity.     

Amino acid residues responsible for the recognition of dichloroacetate by pyruvate dehydrogenase kinase 2

Dichloroacetate (DCA) is a promising anticancer and antidiabetic compound targeting the mitochondrial pyruvate dehydrogenase kinase (PDHK). This study was undertaken in order to map the DCA-binding site of PDHK2. Here, we present evidence that R114, S83, I157 and, to some extent, H115 are essential for DCA binding. We also show that Y80 and D117 are required for the communication between the DCA-binding site and active site of PDHK2. These observations provide important insights into the mechanism of DCA action that may be useful for the design of new, more potent therapeutic compounds.     

Therapeutic potential of dichloroacetate for pyruvate dehydrogenase complex deficiency

We reviewed the use of oral dichloroacetate (DCA) in the treatment of children with congenital lactic acidosis caused by mutations in the pyruvate dehydrogenase complex (PDC). The case histories of 46 subjects were analyzed with regard to diagnosis, clinical presentation and response to DCA. DCA decreased blood and cerebrospinal fluid lactate concentrations, and was generally well tolerated. DCA may be particularly effective in children with PDC deficiency by stimulating residual enzyme activity and, consequently, cellular energy metabolism. A controlled trial is needed to determine the definitive role of DCA in the management of this devastating disease.     

Marked differences between two isoforms of human pyruvate dehydrogenase kinase

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 microm 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.     

Monovalent cation requirement for ADP inhibition of pyruvate dehydrogenase kinase

ADP is a competitive inhibitor with respect to ATP for pyruvate dehydrogenase kinase. Evidence is presented that K⁺ or NH₄⁺ ions are required for inhibition of the kinase by ADP. K⁺ at 30–90 mM and NH₄⁺ at 1–5 mM decrease markedly the apparent K_i of bovine kidney pyruvate dehydrogenase kinase for ADP and also decrease, to a lesser extent, the apparent K_m for ATP. Na⁺ is less effective and, in addition, inhibits kinase activity. Since K⁺ and NH₄⁺ are not required for kinase activity, their effect appears to be primarily of regulatory significance. K⁺ and NH₄⁺ have little effect, if any, on pyruvate dehydrogenase phosphatase activity. When both the kinase and the phosphatase are present and functional, the near steady state activity of the pyruvate dehydrogenase complex is affected significantly by varying the concentration of K⁺ or NH₄⁺ at a fixed ADP/ATP concentration ratio and by varying the $\text{ADP}\text{ATP}$ ratio at a fixed concentration of monovalent cation.     

Synthetic peptide substrates for mammalian pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase

The specificities of pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase were probed using synthetic peptides corresponding to the sequence around phosphorylation sites 1 and 2 on pyruvate dehydrogenase [Tyr-His-Gly-His-Ser(P1)-Met-Ser-Asp-Pro-Gly-Val-Ser(P2)-Tyr-Arg]. The dephosphotetradecapeptide containing aspartic acid at position 8 was a better substrate for the kinase than was the tetradecapeptide containing asparagine at position 8. The apparent Km and V values for the two peptides were 0.43 and 6.1 mM and 2.7 and 2.4 nmol of 32P incorporated/min/mg, respectively. Methylation of the aspartic acid residue also increased the apparent Km of the tetradecapeptide about 14-fold. These results indicate that an acidic residue on the carboxyl-terminal side of phosphorylation site 1 is an important specificity determinant for the kinase. Phosphate was incorporated only into site 1 of the synthetic peptide by the kinase. The phosphatase exhibited an apparent Km of 0.28 mM and a V of 2.3 mumol of 32P released/min/mg for the phosphorylated tetradecapeptide containing aspartic acid. Methylation of the aspartic acid residue had no significant effect on dephosphorylation. The octapeptide and phosphooctapeptide produced by cleavage of the aspartyl-prolyl bond by formic acid were poorer substrates for the kinase and phosphatase than were the tetradecapeptide and phosphotetradecapeptide, respectively. Modification of the amino terminal by acetylation or lysine addition had only a slight effect on the kinase and phosphatase activities.     

Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids

1. Monochloroacetate, dichloroacetate, trichloroacetate, difluoroacetate, 2-chloropropionate, 2,2'-dichloropropionate and 3-chloropropionate were inhibitors of pig heart pyruvate dehydrogenase kinase. Dichloroacetate was also shown to inhibit rat heart pyruvate dehydrogenase kinase. The inhibition was mainly non-competitive with respect to ATP. The concentration required for 50% inhibition was approx. 100mum for the three chloroacetates, difluoroacetate and 2-chloropropionate and 2,2'-dichloropropionate. Dichloroacetamide was not inhibitory. 2. Dichloroacetate had no significant effect on the activity of pyruvate dehydrogenase phosphate phosphatase when this was maximally activated by Ca(2+) and Mg(2+). 3. Dichloroacetate did not increase the catalytic activity of purified pig heart pyruvate dehydrogenase. 4. Dichloroacetate, difluoroacetate, 2-chloropropionate and 2,2'-dichloropropionate increased the proportion of the active (dephosphorylated) form of pyruvate dehydrogenase in rat heart mitochondria with 2-oxoglutarate and malate as respiratory substrates. Similar effects of dichloroacetate were shown with kidney and fat-cell mitochondria. Glyoxylate, monochloroacetate and dichloroacetamide were inactive. 5. Dichloroacetate increased the proportion of active pyruvate dehydrogenase in the perfused rat heart, isolated rat diaphragm and rat epididymal fat-pads. Difluoroacetate and dichloroacetamide were also active in the perfused heart, but glyoxylate, monochloroacetate and trichloroacetate were inactive. 6. Injection of dichloroacetate into rats starved overnight led within 60 min to activation of pyruvate dehydrogenase in extracts from heart, psoas muscle, adipose tissue, kidney and liver. The blood concentration of lactate fell within 15 min to reach a minimum after 60 min. The blood concentration of glucose fell after 90 min and reached a minimum after 120 min. There was no significant change in plasma glycerol concentration. 7. In epididymal fatpads dichloroacetate inhibited incorporation of (14)C from [U-(14)C]glucose, [U-(14)C]fructose and from [U-(14)C]lactate into CO(2) and glyceride fatty acid. 8. It is concluded that the inhibition of pyruvate dehydrogenase kinase by dichloroacetate may account for the activation of pyruvate dehydrogenase and pyruvate oxidation which it induces in isolated rat heart and diaphragm muscles, subject to certain assumptions as to the distribution of dichloroacetate across the plasma membrane and the mitochondrial membrane. 9. It is suggested that activation of pyruvate dehydrogenase by dichloroacetate could contribute to its hypoglycaemic effect by interruption of the Cori and alanine cycles. 10. It is suggested that the inhibitory effect of dichloroacetate on fatty acid synthesis in adipose tissue may involve an additional effect or effects of the compound.     

Peptides derived from pyruvate dehydrogenase as substrates for pyruvate dehydrogenase kinase and phosphatase.

Evidence is presented that phosphopeptides produced by tryptic digestion of phosphorylated pyruvate dehydrogenase are effective substrates for pyruvate dehydrogenase phosphatase and that the dephosphopeptides can serve as substrates for pyruvate dehydrogenase kinase. These findings indicate that the phosphatase and the kinase do not require an intact tertiary structure in pyruvate dehydrogenase, but apparently recognize components of the local primary sequence around the phosphorylation sites.     

The inhibition of lactate dehydrogenase by hydrated pyruvate

The lactate dehydrogenase-catalyzed reduction of pyruvate by NADH was studied using a spectroscopic method. The inhibitory effect exhibited by high concentrations of pyruvate was investigated in phosphate and 2,2-diethylmalonate buffers. Kinetic studies were carried out in which the rate of the enzyme-catalyzed reaction was monitored at various stages of pyruvate hydration, H₂O + CH₃COCO₂^? ? CH₃C(OH)₂2C0₂^?. Buffered solutions of different initial relative amounts of ketopyruvate and hydrated pyruvate (2,2-dihydroxypropanoic acid) were also preincubated with the enzyme and NAD⁺. Kinetic runs were initiated in the resultant solutions at various stages of incubation by the introduction of NADH. The results of the present investigation indicate that hydrated pyruvate is a major inhibitor of lactate dehydrogenase and forms an inhibitory complex with the enzyme and oxidized coenzyme.     

Activation of pyruvate dehydrogenase in perfused rat heart by dichloroacetate (Short Communication)

The activity of pyruvate dehydrogenase was assayed in extracts of rat hearts perfused in vitro with media containing glucose and insulin±acetate±dichloroacetate. Dichloroacetate (100μm, 1mm or 10mm) increased the activity of pyruvate dehydrogenase in perfusions with glucose or glucose+acetate. Evidence is given that dichloroacetate may facilitate the conversion of pyruvate dehydrogenase from an inactive (phosphorylated) form into an active (dephosphorylated) form.     

Effects of alpha-ketoisovalerate on bovine heart pyruvate dehydrogenase complex and pyruvate dehydrogenase kinase

The regulatory effects of alpha-ketoisovalerate on purified bovine heart pyruvate dehydrogenase complex and endogenous pyruvate dehydrogenase kinase were investigated. Incubation of pyruvate dehydrogenase complex with 0.125 to 10 mM alpha-ketoisovalerate caused an initial lag in enzymatic activity, followed by a more linear but inhibited rate of NADH production. Incubation with 0.0125 or 0.05 mM alpha-ketoisovalerate caused pyruvate dehydrogenase inhibition, but did not cause the initial lag in pyruvate dehydrogenase activity. Gel electrophoresis and fluorography demonstrated the incorporation of acyl groups from alpha-keto[2-14C]isovalerate into the dihydrolipoyl transacetylase component of the enzyme complex. Acylation was prevented by pyruvate and by arsenite plus NADH. Endogenous pyruvate dehydrogenase kinase activity was stimulated specifically by K+, in contrast to previous reports, and kinase stimulation by K+ correlated with pyruvate dehydrogenase inactivation. Maximum kinase activity in the presence of K+ was inhibited 62% by 0.1 mM thiamin pyrophosphate, but was inhibited only 27% in the presence of 0.1 mM thiamin pyrophosphate and 0.1 mM alpha-ketoisovalerate. Pyruvate did not affect kinase inhibition by thiamin pyrophosphate at either 0.05 or 2 mM. The present study demonstrates that alpha-ketoisovalerate acylates heart pyruvate dehydrogenase complex and suggests that acylation prevents thiamin pyrophosphate-mediated kinase inhibition.     

Characterization of the inhibition of Escherichia coli pyruvate dehydrogenase complex by pyruvate

The E. coli pyruvate dehydrogenase complex was inhibited by pyruvate in absence of its cofactor, NAD+. The inhibition was found to increase with pH and phosphate concentration of the buffer and decrease with its ionic strength. The inhibition profile was different with MOPS buffer. No radioactivity was found in the enzyme, when the latter was incubated with 2-14C-pyruvate. The results suggest that covalent adduct formation is not necessary for the observed inhibition.     

Distinct mechanisms for dysfunctions of mutated ryanodine receptor isoforms

Ryanodine receptor (RyR) is the Ca²⁺-induced Ca²⁺ release channel in cells. RyR1 and RyR2 are its isoforms expressed in the skeletal and cardiac muscles, respectively. Their missense mutations, which are clustered in three regions that correspond to each other, cause hereditary disorders such as malignant hyperthermia and central core disease in skeletal muscle and catecholaminergic polymorphic ventricular tachycardia in cardiac muscle. Their pathogeneses, however, are not well understood. The following hypotheses are favorably discussed in this article: phenotypes with RyR1 and RyR2 mutations are mainly caused by dysregulations of their functions through the interdomain interaction and luminal Ca²⁺, respectively.     

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

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.     

Apparent inhibition of pyruvate kinase by phosphocreatine and phosphoarginine.

1. Addition of a non-dialysable, heat-labile and acid-precipitable factor which was not absorbed on DEAE-cellulose column, could restore the sensitivity of the chromatographed muscle pyruvate kinase from Marphysa sanguinea towards phosphocreatine inhibition. 2. This factor, being non-specific as it acts on pyruvate kinase isozymes from different sources, demonstrated high creatine kinase activity. 3. High concentrations of ADP, creatine or replacement of ADP with IDP/UDP or high pH abolished the inhibition indicating that the inhibition was mediated through creatine kinase by depleting ADP. 4. Apparent inhibition of phosphocreatine was related to the relative activities of 3 intracellular enzymes--pyruvate kinase, creatine kinase and adenosine triphosphatase.     

Structural determinants for cross-talk between pyruvate dehydrogenase kinase 3 and lipoyl domain 2 of the human pyruvate dehydrogenase complex

Pyruvate dehydrogenase kinase isoforms (PDK1-4) are the molecular switch that down-regulates activity of the human pyruvate dehydrogenase complex through reversible phosphorylation. We showed previously that binding of the lipoyl domain 2 (L2) of the pyruvate dehydrogenase complex to PDK3 induces a "cross-tail" conformation in PDK3, resulting in an opening of the active site cleft and the stimulation of kinase activity. In the present study, we report that alanine substitutions of Leu-140, Glu-170, and Glu-179 in L2 markedly reduce binding affinities of these L2 mutants for PDK3. Unlike wildtype L2, binding of these L2 mutants to PDK3 does not preferentially reduce the affinity of PDK3 for ADP over ATP. The inefficient removal of product inhibition associated with ADP accounts for the decreased stimulation of PDK3 activity by these L2 variants. Serial truncations of the PDK3 C-terminal tail region either impede or abolish the binding of wild-type L2 to the PDK3 mutants, resulting in the reduction or absence of L2-enhanced kinase activity. Alanine substitutions of residues Leu-27, Phe-32, Phe-35, and Phe-48 in the lipoyl-binding pocket of PDK3 similarly nullify L2 binding and L2-stimulated PDK3 activity. Our results indicate that the above residues in L2 and residues in the C-terminal region and the lipoyl-binding pocket of PDK3 are critical determinants for the cross-talk between L2 and PDK3, which up-regulates PDK3 activity.