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Pyruvate dehydrogenase deficiency may be a non-specific consequence of many different neurological degenerative disorders. There are also serious methodological problems in estimating the activity of this enzyme complex.
Protein-protein interactions play an important role in the regulation of enzymic activity of pyruvate dehydrogenase kinase (PDK). It is generally believed that the binding of PDK to the inner lipoyl-bearing domain L2 of the transacetylase component E2 of pyruvate dehydrogenase complex largely determines the level of kinase activity. In the present study, we characterized the interaction between the individual isoenzymes of PDK (PDK1-PDK4) and monomeric L2 domain of human E2, as well as the effect of this interaction on kinase activity. It was found that PDK isoenzymes are markedly different with respect to their affinities for L2. PDK3 demonstrated a very tight binding, which persisted during isolation of PDK3-L2 complexes using size-exclusion chromatography. Binding of PDK1 and PDK2 was readily reversible with the apparent dissociation constant of approx. 10 microM for both isoenzymes. PDK4 had a greatly reduced capacity for L2 binding (relative order PDK3>PDK1=PDK2>PDK4). Monomeric L2 domain alone had very little effect on the activities of either PDK1 or PDK2. In contrast, L2 caused a 3-fold increase in PDK3 activity and approx. 37% increase in PDK4 activity. These results strongly suggest that the interactions between the individual isoenzymes of PDK and L2 domain are isoenzyme-specific and might be among the major factors that determine the level of kinase activity of particular isoenzyme towards the pyruvate dehydrogenase complex.
Pyruvate dehydrogenase complex (PDC) deficiency is an inborn metabolic disorder associated with a variety of neurologic abnormalities. This report describes the development and initial characterization of a novel murine model system in which PDC deficiency has been introduced specifically into the developing nervous system. The absence of liveborn male and a roughly 50% reduction in female offspring following induction of the X-linked mutation indicate that extensive deficiency of PDC in the nervous system leads to pre-natal lethality. Brain tissue from surviving females at post-natal days 15 and 35 was shown to have approximately 75% of wild-type PDC activity, suggesting that a threshold of enzyme activity exists for post-natal survival. Detailed histological analyses of brain tissue revealed structural defects such as disordered neuronal cytoarchitecture and neuropil fibers in grey matter, and reduced size of bundles and disorganization of fibers in white matter. Many of the histologic abnormalities resemble those found in human female patients who carry mutations in the X-linked ortholog. These findings demonstrate a requirement for PDC activity within the nervous system for survival in utero and suggest that impaired pyruvate metabolism in the developing brain can affect neuronal migration, axonal growth and cell-cell interactions.
A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5'end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191, 337-446], was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, Mr 46,265) and 469 amino acids (E3, Mr 49,193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-oxo-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.
The kinase-activator protein (KAP) of pyruvate dehydrogenase complex (PDC) has been purified approx. 2250-fold from high-speed supernatants of mitochondrial extracts from the liver of 48 h-starved rats. Purified KAP demonstrates kinase activity towards both the E1 component of PDC and towards a synthetic peptide corresponding to the major phosphorylation site on E1. Furthermore, the activities of KAP and PDC kinase co-fractionate through several stages of purification and have the same apparent mass. We conclude that KAP is not a distinct protein, but is kinase which has dissociated from the complex.
A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.
Pyruvate dehydrogenase complex (PDHC) is an intramitochondrial multienzyme complex essential for the aerobic oxidation of glucose. The majority of patients with PDHC deficiency have abnormalities in the major catalytic and regulatory subunit, E1 alpha, which is encoded on the X chromosome. The clinical spectrum of PDHC deficiency is heterogeneous, particularly in heterozygous females, and diagnosis may be difficult. Three affected infant girls with PDHC deficiency were investigated. All had dysmorphic features, microcephaly with profound global developmental delay, and hypotonia. Systemic acidosis was absent, although serum lactate and pyruvate were abnormally elevated. Magnetic resonance imaging revealed hypoplasia of the corpus callosum in all patients. Proton magnetic resonance spectroscopy of brain revealed large increases in relative signal intensities for lactic acid and decreases in the relative signal intensities of N-acetylaspartate, a marker of neuronal damage or less. Phosphorus MRS of muscle revealed abnormally low phosphorylation potentials for all these patients, although the degree of abnormality was variable and not directly correlated with the amount of brain lactate. It is proposed that cerebral dysgenesis and cerebral lactic acidemia as shown by magnetic resonance imaging and proton magnetic resonance spectroscopy are useful diagnostic clues to PDHC deficiency, particularly in females in whom variable patterns of X-inactivation reduce sensitivity of laboratory diagnosis based on the biochemical studies of peripheral tissues. In addition, muscle bioenergetic abnormalities in conjunction with CNS dysfunction may contribute to profound hypotonia in this disorder.
Mammalian pyruvate dehydrogenase actively catalyzed the oxidation of methylglyoxal to acetyl-CoA. The reaction was fully enzymatic with an estimated Km of 1.89 mM. On the other hand, methylglyoxal was a competitive inhibitor of the enzyme for pyruvate, the Ki being in the 1 mM range. The reaction was inhibited in the presence of HgCl2. The reaction products were quantitatively identified as acetyl-CoA and formic acid. A mechanism for the reaction is proposed.
Pyruvate dehydrogenase complex was purified from rat heart. A new component(mol.wt; 52,000) was found in the purified complex in addition to well known three component enzymes. This component(referred to as component X) was acetylated with [2-14C] pyruvate in the absence of CoA as well as lipoate acetyltransferase. The anti-lipoate acetyltransferase antibody reacted with component X and lipoate acetyltransferase, suggesting that component X shows homology with lipoate acetyltransferase in protein structure. cDNA for lipoate acetyltransferase was isolated from rat liver cDNA library in lambda gt 11. cDNA for lipoate acetyltransferase recognized two kinds of mRNAs of 3.5 Kb and 2.5 Kb.
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Pyruvate dehydrogenase complex (PDC) has a key role in the regulation of hepatic lipogenesis by dietary factors. We have investigated the effects of dietary carbohydrate and fat on hepatic PDC. Sucrose-based or starch-based diets were administered for 15 days. A positive correlation between PDC activity and the lipogenic potential of the diet was found. A high-sucrose, fat-free diet caused a 3-fold increase in total activity whereas a high-starch, fat-free diet caused a 1.5-fold increase, as compared with chow-fed rats. Dietary polyunsaturated fat (PUF) caused a marked inhibitory effect on total and active PDC; fish oil being more effective than corn oil. Dietary saturated fat (butter) failed to inhibit the sucrose-induced elevation in total activity, but was almost as effective as fish oil in depressing percent active enzyme. Changes in total PDC activity closely correlated with modifications in the content of enzyme quantitated by immunoblotting, indicating that increased enzyme content and not activation is the predominant mechanism underlying the adaptive response to high-sucrose feeding. This response is suppressed by dietary PUF. Inhibition of hepatic lipogenesis by PUF involves a reduction of PDC content as well as that of several lipogenic enzymes. The relevant mechanisms remain to be established.
A 20-bp deletion in the last exon of the pyruvate dehydrogenase (PDH) E1 alpha gene was found in a severely affected female patient diagnosed with PDH deficiency. PDH-complex activity in the patient's fibroblasts was 22% of that in normal controls. The mutation was characterized using PCR techniques with both patient cDNA and genomic DNA, followed by sequencing of the products. E1 beta cDNA sequence was found to be the same as that in controls. The deletion causes a frameshift and the occurrence of a premature stop codon. Western blot analysis revealed an extra band migrating just above the PDH E1 beta band. Northern blot analysis showed normal levels of both E1 alpha and E1 beta message when probed with the respective cDNAs. However, a larger intermediate-size transcript was observed for this patient in the E1 beta blot. The 20-bp deletion was not found in either parent's genomic DNA, and hence we conclude that the mutation must have occurred de novo, either in the germ-line cells or immediately following fertilization.
The pyruvate dehydrogenase complex has a central role in the regulation of mammalian metabolism as it represents the point-of-no-return in the utilization of carbohydrate. This article summarizes our studies into how signalling systems initiated by hormones binding to cell surface receptors can reach the pyruvate dehydrogenase system which is located within the inner mitochondrial membrane. One class of hormones which activate pyruvate dehydrogenase are those that increase cytoplasmic Ca2+. A wide range of studies on isolated enzymes, separated mitochondria and intact cell preparations have shown that the activation is due to the stimulation of pyruvate dehydrogenase phosphatase. Two other intramitochondrial dehydrogenases which regulate the citrate acid cycle are activated in parallel and this is an important means of balancing the supply of ATP to increasing cell demand. Insulin is also able to activate pyruvate dehydrogenase, but this is restricted to fat and other cells capable of lipogenesis. Insulin acts by stimulating pyruvate dehydrogenase phosphatase, but the activation does not involve alterations in Ca2+. The signalling pathway involved has not been established, but it appears to be quite distinct from those involved in many other actions of insulin.
The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.
The pyruvate dehydrogenase kinase-catalyzed inactivation of the pyruvate dehydrogenase complex was studied using dialyzed, soluble proteins from mitochondria purified from green leaf tissue of Pisum sativum L. seedlings. At subsaturating ATP concentrations, K+ or NH4+, but not Na+, stimulated the pyruvate dehydrogenase kinase by lowering the Km(ATP). Micromolar concentrations of NH4+ were required to produce the same effect as millimolar concentrations of K+. This is apparent from the observations that the activation constant (Kact) for NH4+ was 0.1 mM, whereas the Kact(K+) was 0.7 mM. Maximal pyruvate dehydrogenase kinase velocities attained with NH4+ were higher than those with K+, and, therefore, NH4+ was able to stimulate PDH kinase further in the presence of saturating K+. This result supports our conclusion that photorespiratory NH4+ production in plant mitochondria may be involved in regulating the entry of carbon into the Krebs cycle by way of the pyruvate dehydrogenase complex.
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The proportion of pyruvate dehydrogenase (PDH) complex in the active dephosphorylated form was decreased (compared with fed lean control mice) in heart muscle mitochondria after the induction of obesity with gold-thioglucose (by 54%) or starvation of lean mice for 48 h (by 81%). The effects of obesity to inactivate PDH complex were demonstrable 4 weeks after administration of gold-thioglucose, and occurred despite significant hyperinsulinaemia in obese animals. Phosphorylation and inactivation of PDH complex in mouse heart muscle in starvation was attributed to a stable increase (2.7-fold) in the activity of PDH kinase as measured in extracts of mitochondria mediated by increased specific activity of a protein activator of PDH kinase (KAP) [Denyer, Kerbey & Randle (1986) Biochem. J. 239, 347-354]. In obese mice no such increase in kinase activity was observed, and we conclude that phosphorylation and inactivation of PDH complex in heart muscle in obesity is not mediated by KAP, but rather is a consequence of increased lipid oxidation.
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