Mutations in the X-linked E1α subunit of pyruvate dehydrogenase leading to deficiency of the pyruvate dehydrogenase complex

Chun, Kathy; MacKay, N.; Petrova‐Benedict, Roumyana; Robinson, Brian H.

doi:10.1093/hmg/2.4.449

Cited by 70 publications

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“…Two patients with F176L mutation had decreased activity (about 25% of the control cells) and reduced amount of PDH1 protein (41,42). ␣Phe-176 in the structure of PDH1 is close to ␣ЈPhe-176 (3.7 Å distance between the rings), the same residue in the neighboring ␣Ј subunit (Fig.…”

Section: Discussionmentioning

confidence: 93%

“…Serines (S) of the three phosphorylation sites are shown in red and identified as sites 1-3 in red above the serines. Nonidentical residues are in the colored boxes as follows: yellow indicates homologous substitutions; orange indicates nonhomologous substitutions; pink indicates His-15 and Phe-176 of PDH1, the replacement of which is known to cause PDH deficiency (38,41,42); and green indicates substitutions involving prolines in the C-terminal region. The TPP motif sequences are underlined with a single line, and the C-terminal region with multiple proline replacement is identified by doubled underlines.…”

Section: Sequence Comparison Between ␣ Subunits Of Pdh2 and Pdh1-mentioning

confidence: 99%

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Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Korotchkina

Sidhu

Patel

2006

Journal of Biological Chemistry

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Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the ␣ subunit of ␣ 2 ␤ 2 PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37°C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 ␣ subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1. Mammalian pyruvate dehydrogenase complex (PDC)2 plays a central role in glucose oxidation by catalyzing the oxidative decarboxylation of pyruvic acid with formation of carbon dioxide, acetyl-CoA, NADH, and H ϩ (for reviews see Refs. 1-3). PDC is composed of multiple copies of the following three catalytic components: (i) pyruvate dehydrogenase (PDH) catalyzing the decarboxylation of pyruvate and reductive acetylation of the lipoyl moieties of dihydrolipoamide acetyltransferase (E2); (ii) E2 transferring acetyl moiety to CoA; (iii) and dihydrolipoamide dehydrogenase (E3) reoxidizing the reduced lipoyl moieties of E2 with the reduction of NAD ϩ to NADH (1-3). Forty eight to 60 subunits of E2and 12 subunits of E3-binding protein (BP), a noncatalytic component of mammalian PDC, form the central core of the multienzyme PDC, which binds 20 -30 tetramers of PDH, 6 -12 dimers of E3 and two regulatory enzymes, 1-2 copies of pyruvate dehydrogenase kinase (PDK), and 2-3 copies of pyruvate dehydrogenase phosphatase (PDP) (4, 5). PDK and PDP provide the finely tuned regulation of activity of PDC through reversible phosphorylation-dephosphorylation of PDH (and hence PDC) depending on cellular demand for glucose oxidation. Mammalian PDH is a tetramer consisting of two ␣ and two ␤ subunits. The hum...

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Section: Discussionmentioning

confidence: 93%

Section: Sequence Comparison Between ␣ Subunits Of Pdh2 and Pdh1-mentioning

confidence: 99%

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Korotchkina

Sidhu

Patel

2006

Journal of Biological Chemistry

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“…Since the S293A mutant E1 is active and does not show an altered K m for thiamine pyrophosphate, it seems likely that the negative charge introduced by mutating serine 293 to glutamate may neutralize the ability of histidine 292 to interact with thiamine pyrophosphate. That the current structural models for thiamine pyrophosphate binding sites may also apply to pyruvate dehydrogenases was demonstrated by the affect of one of the reported human genetic defects of PDH (26). The defect was found to be the result of a naturally occurring leucine substitution of the conserved histidine residue, adjacent to the phosphorylation site 1 serine residue, in the thiamine pyrophosphate binding motif of PDH E1␣ (26).…”

Section: Discussionmentioning

confidence: 99%

“…That the current structural models for thiamine pyrophosphate binding sites may also apply to pyruvate dehydrogenases was demonstrated by the affect of one of the reported human genetic defects of PDH (26). The defect was found to be the result of a naturally occurring leucine substitution of the conserved histidine residue, adjacent to the phosphorylation site 1 serine residue, in the thiamine pyrophosphate binding motif of PDH E1␣ (26). This leucine substitution, in a heterozygous individual, resulted in less than 2% of normal PDH activity compared to controls (26).…”

Section: Discussionmentioning

confidence: 99%

“…The defect was found to be the result of a naturally occurring leucine substitution of the conserved histidine residue, adjacent to the phosphorylation site 1 serine residue, in the thiamine pyrophosphate binding motif of PDH E1␣ (26). This leucine substitution, in a heterozygous individual, resulted in less than 2% of normal PDH activity compared to controls (26). However, direct evidence of an actual role of this conserved residue in thiamine pyrophosphate binding by ␣-ketoacid dehydrogenases has not previously been provided.…”

Section: Discussionmentioning

confidence: 99%

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Roles of Amino Acid Residues Surrounding Phosphorylation Site 1 of Branched-chain α-Ketoacid Dehydrogenase (BCKDH) in Catalysis and Phosphorylation Site Recognition by BCKDH Kinase

Hawes

Schnepf

Jenkins

et al. 1995

Journal of Biological Chemistry

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Branched-chain ␣-ketoacid dehydrogenase is regulated by reversible phosphorylation of serine 293 (site 1) on the E1␣ subunit. Alanine-scanning mutagenesis was used to examine the roles of residues surrounding serine 293 in catalysis by the dehydrogenase and in substrate recognition by branched-chain ␣-ketoacid dehydrogenase kinase. Alanine substitution of serine 293 resulted in a 10-fold increased K m for ␣-ketoisovalerate, a less increased (2.8-fold) K m for ␣-ketoisocaproate, but no change in V max or the K m for thiamine pyrophosphate. Alanine substitutions of arginine 288, histidine 292, and aspartate 296, residues highly conserved among ␣-ketoacid dehydrogenases, resulted in inactive enzymes. Each of the inactive E1 mutants bound to the E2 core subunit with equal affinity as wild-type E1, and each produced circular dichroism spectra identical to that of wild-type E1. Two mutations, H292A and S293E, abolished the ability of E1 apoenzyme to reconstitute with thiamine pyrophosphate. Each alanine-substituted E1 was phosphorylated at site 1 by branched-chain ␣-ketoacid dehydrogenase kinase with similar rates, with the exception of the R288A mutant, which displayed no detectable phosphorylation. Thiamine pyrophosphate inhibited the phosphorylation of all mutant enzymes with the exception of H292A, the mutant E1 that did not bind thiamine pyrophosphate.The activity state of mammalian branched-chain ␣-ketoacid dehydrogenase (BCKDH) 1 is highly regulated according to the physiological requirements for either degradation or conservation of branched-chain amino acids derived from cellular and dietary protein (1, 2). This regulation is primarily exerted through reversible phosphorylation of the E1␣ subunit by BCKDH kinase. BCKDH kinase phosphorylates two serine residues on the E1␣ subunit, although inactivation of the dehydrogenase results exclusively from the phosphorylation of site 1, serine 293 in rat E1␣ (3-5). Furthermore, phosphorylation of site 2 occurs at a significantly slower rate (3-5), suggesting that the site 2 serine, serine 303 in rat E1␣, is a poor substrate either due to the primary or secondary structure surrounding this residue or the positioning of this serine residue relative to the kinase active site. BCKDH kinase is regulated in part through inhibition by branched-chain ␣-ketoacids and thiamine pyrophosphate, all of which are substrates for the BCKDH-catalyzed reaction (6, 7). Inhibition of the kinase by branched-chain ␣-ketoacids is believed to occur in vivo, resulting in a fully active BCKDH in the liver of rats fed a high protein diet (2). However, the exact molecular basis for these inhibitory effects is not clearly understood. BCKDH kinase has also been reported to be activated by its interaction with the E2 subunit of the BCKDH complex, although the molecular basis for this mode of regulation is also not understood (8, 9). Another important feature of BCKDH kinase is its high degree of substrate specificity, resulting in phosphorylation only of BCKDH E1␣ but not the highly homologous E1␣ su...

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Mutation analysis of the pyruvate dehydrogenase E1α gene in eight patients with a pyruvate dehydrogenase complex deficiency

Lissens¹,

Meırleır²,

Seneca³

et al. 1996

Hum. Mutat.

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Most of the mutations causing deficiency of the pyruvate dehydrogenase (PDH) complex are in the X-linked E ^ gene. We have developed a rapid screening method for the detection of mutations in this gene using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and single-strand conformation polymorphism (SSCP) analysis. With this method, we studied eight patients with a PDH complex deficiency, using cultured fibroblasts. In all patients, aberrant SSCP patterns were found and, after sequencing of the corresponding fragments, we were able to identify six new mutations and two mutations already described previously. The mutations are point mutations leading to amino acid substitutions (5) and direct repeat insertions (3). The presence of the mutations was confirmed in genomic fibroblast DNA* The 4 female patients were shown to carry both a normal and a mutated Exa gene.

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Mutations in the X-linked E1α subunit of pyruvate dehydrogenase leading to deficiency of the pyruvate dehydrogenase complex

Cited by 70 publications

References 0 publications

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Roles of Amino Acid Residues Surrounding Phosphorylation Site 1 of Branched-chain α-Ketoacid Dehydrogenase (BCKDH) in Catalysis and Phosphorylation Site Recognition by BCKDH Kinase

Mutation analysis of the pyruvate dehydrogenase E1α gene in eight patients with a pyruvate dehydrogenase complex deficiency

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