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{{STRUCTURE_1lvl | PDB=1lvl | SCENE= }} | {{STRUCTURE_1lvl | PDB=1lvl | SCENE= }} | ||
==General== | ==General Structure and Information== | ||
Dihydrolipoamide dehydrogenase(E3), a component of the Saccharomyces cerevisiae and mammalian [[Pyruvate dehydrogenase]] complexes (PDC), anchors an E3 homodimer inside each of the 12 pentagonal faces of the 60-mer dihydrolipoamide acetyltransferase (E2)[http://pubs.acs.org/doi/full/10.1021/bi9600254?prevSearch=%2528Dihydrolipoamide%2Bdehydrogenase%2B%2528E3%2529%2529%2BNOT%2B%255Batype%253A%2Bad%255D%2BNOT%2B%255Batype%253A%2Bacs-toc%255D&searchHistoryKey=] PDC is the enzyme in the citric acid cycle responsible for the reaction converting Pyruvate to Acetyl CoA, NAD+ to NADH and H+ and the release of carbon dioxide. | Dihydrolipoamide dehydrogenase(E3), a component of the Saccharomyces cerevisiae and mammalian [[Pyruvate dehydrogenase]] complexes (PDC), anchors an E3 homodimer inside each of the 12 pentagonal faces of the 60-mer dihydrolipoamide acetyltransferase (E2)[http://pubs.acs.org/doi/full/10.1021/bi9600254?prevSearch=%2528Dihydrolipoamide%2Bdehydrogenase%2B%2528E3%2529%2529%2BNOT%2B%255Batype%253A%2Bad%255D%2BNOT%2B%255Batype%253A%2Bacs-toc%255D&searchHistoryKey=] PDC is the enzyme in the citric acid cycle responsible for the reaction converting Pyruvate to Acetyl CoA, NAD+ to NADH and H+ and the release of carbon dioxide. E3's cofactors include NAD+ and FAD, and unlike E1 and E2, there are equal subunits noncovalently bonded to the 60-meric transacetylase core in both prokaryotes and eukaryotes. The jmol image shown upon loading the page is the base subunit which is bonded to the core. | ||
E3 is common to all a-ketoacid dehydrogenase complexes. Errors in the gene coding human E3 cause combined deficiencies in a-ketoacid dehydrogenase complexes manifested by lactic acidemias and Maple Syrup Urine Disease[http://en.wikipedia.org/wiki/Maple_syrup_urine_disease]. A subset of the human E3 mutations has been suggested to occur at the homodimer interface or at the putative E3/E3BP interaction surface | E3 is common to all a-ketoacid dehydrogenase complexes. Errors in the gene coding human E3 cause combined deficiencies in a-ketoacid dehydrogenase complexes manifested by lactic acidemias and Maple Syrup Urine Disease[http://en.wikipedia.org/wiki/Maple_syrup_urine_disease]. A subset of the human E3 mutations has been suggested to occur at the homodimer interface or at the putative E3/E3BP interaction surface | ||
[[Image: pyruvate_dehydrogenase.gif|500px|left|thumb]] | [[Image: pyruvate_dehydrogenase.gif|500px|left|thumb]] | ||
==Structure== | E3 is synthesized as a precursor form in the cytoplasm and imported into mitochondria, and the mature E3 migrates as a 55 kDa polypeptide in SDS-PAGE though its calculated molecular mass with one molecule of FAD is 51 kDa. | ||
==Structure in Pyruvate Dehydrogenase Complex== | |||
In ''E. coli'' this complex exists as 24 E2 proteins arranged in a cube, surrounded by 12 E1 proteins and 12 E3 proteins. Dihidrolipoamide dehydrogenase (E3) binds to the pyruvate dehydrogenase complex (and the center of the cube of E2 proteins) through a <scene name='Nicholas_Rockefeller_Sandbox/E3_plus_e3_binding_protein/1'>coupling with E3 binding protein</scene>.‘<ref>PMID:16442803</ref>’ The E3 binding protein is a completely separate protein from E3, but serves to connect the E3 polypeptides to the overarching structure. The active site includes an FAD group, as well as <scene name='Nicholas_Rockefeller_Sandbox/Cys43cys48/3'>Cys 43 and Cys 48</scene> forming a disulfide bond. When the substrate is not present, <scene name='Nicholas_Rockefeller_Sandbox/Tyr181/2'>Tyr 181</scene> “covers” the catalytic site from being exposed to solvents. Dihidrolipoamide dehydrogenase (E3) is a SCOP alpha and beta (a/b) class protein of the FAD/NAD(P)-binding domain fold. | In ''E. coli'' this complex exists as 24 E2 proteins arranged in a cube, surrounded by 12 E1 proteins and 12 E3 proteins. Dihidrolipoamide dehydrogenase (E3) binds to the pyruvate dehydrogenase complex (and the center of the cube of E2 proteins) through a <scene name='Nicholas_Rockefeller_Sandbox/E3_plus_e3_binding_protein/1'>coupling with E3 binding protein</scene>.‘<ref>PMID:16442803</ref>’ The E3 binding protein is a completely separate protein from E3, but serves to connect the E3 polypeptides to the overarching structure. The active site includes an FAD group, as well as <scene name='Nicholas_Rockefeller_Sandbox/Cys43cys48/3'>Cys 43 and Cys 48</scene> forming a disulfide bond. When the substrate is not present, <scene name='Nicholas_Rockefeller_Sandbox/Tyr181/2'>Tyr 181</scene> “covers” the catalytic site from being exposed to solvents. Dihidrolipoamide dehydrogenase (E3) is a SCOP alpha and beta (a/b) class protein of the FAD/NAD(P)-binding domain fold. | ||
==Mechanism== | ==Mechanism== | ||
The redox reaction occurs through the influence of <scene name='Nicholas_Rockefeller_Sandbox/Cys43cys48/3'>Cys 43 and Cys 48</scene>, between which there is a disulfide bond within a distorted alpha helix. This redox active disulfide bond becomes reduced in order to reoxidize the E2 enzyme of the multienzyme complex.‘<ref>Voet, Donald et al. 2008. Fundamentals of Biochemistry. 3rd ed. pp.570-575</ref>’ E2 donates protons and electrons to E3 in order to complete its catalytic cycle. The E3 enzyme’s flavin ring (FAD) funnels electrons from the disulfide bond to itself, <scene name='Nicholas_Rockefeller_Sandbox/Fadnad/1'>then binds to NAD+ and donates electrons</scene>, reoxidizing the E3, and leaving it ready for the beginning of its catalytic cycle again. | The redox reaction occurs through the influence of <scene name='Nicholas_Rockefeller_Sandbox/Cys43cys48/3'>Cys 43 and Cys 48</scene>, between which there is a disulfide bond within a distorted alpha helix. This redox active disulfide bond becomes reduced in order to reoxidize the E2 enzyme of the multienzyme complex.‘<ref>Voet, Donald et al. 2008. Fundamentals of Biochemistry. 3rd ed. pp.570-575</ref>’ E2 donates protons and electrons to E3 in order to complete its catalytic cycle. The E3 enzyme’s flavin ring (FAD) funnels electrons from the disulfide bond to itself, <scene name='Nicholas_Rockefeller_Sandbox/Fadnad/1'>, oxidizing dihydrolipoate to its lipoate resting state and producing FADH2, then binds to the NAD+ cofactor and donates electrons</scene>, reoxidizing the E3, regenerating FAD, producing NADH, and leaving it ready for the beginning of its catalytic cycle again. | ||
[[Image: PyruvateDehydrgenaseMech1.gif|1000px|left|thumb]] | |||
A 16-amino acid leader sequence addition changes the kinetic mechanism of human E3 so that it resembles the mixed sequential and ping-pong mechanism of [[Glutathione Reductase]]‘<ref>Kim, Hakjung Expression of cDNA Sequences Encoding Mature and Precursor Forms of Human Dihydrolipoamide Dehydrogenase in Escherichia coli. Journal of Biological Chemistry. 266, 15. 1991.</ref>’ | |||
==Regulation== | ==Regulation== | ||
The regulation of | The regulation of E3 kinetically comes through regulation of the entire Pyruvate Dehydrogenase complex. As would be expected, one of the main regulators is the presence of its product, acetyl-CoA as well as NADH. NADH competes with NAD+ with the binding site on E3, therefore regulating the entire pyruvate dehydrogenase complex when NADH is in high concentration.‘<ref>Voet, Donald et al. 2008. Fundamentals of Biochemistry. 3rd ed. p.585</ref>’ | ||