Information on EC 2.3.1.12 - dihydrolipoyllysine-residue acetyltransferase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
2.3.1.12
-
RECOMMENDED NAME
GeneOntology No.
dihydrolipoyllysine-residue acetyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + enzyme N6-(dihydrolipoyl)lysine = CoA + enzyme N6-(S-acetyldihydrolipoyl)lysine
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
acetyl CoA biosynthesis
-
-
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
-
-
Citrate cycle (TCA cycle)
-
-
Glycolysis / Gluconeogenesis
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
NIL
-
-
oxidative decarboxylation of pyruvate
-
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pyruvate decarboxylation to acetyl CoA
-
-
Pyruvate metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:enzyme N6-(dihydrolipoyl)lysine S-acetyltransferase
A multimer (24-mer or 60-mer, depending on the source) of this enzyme forms the core of the pyruvate dehydrogenase multienzyme complex, and binds tightly both EC 1.2.4.1, pyruvate dehydrogenase (acetyl-transferring) and EC 1.8.1.4, dihydrolipoyl dehydrogenase. The lipoyl group of this enzyme is reductively acetylated by EC 1.2.4.1, and the only observed direction catalysed by EC 2.3.1.12 is that where the acetyl group is passed to coenzyme A.
CAS REGISTRY NUMBER
COMMENTARY hide
9032-29-5
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
broccoli
-
-
Manually annotated by BRENDA team
cauliflower
-
-
Manually annotated by BRENDA team
pigeon
-
-
Manually annotated by BRENDA team
Hansenula miso
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
cell lines BML1-ME, NIH3T3, PA317 and PE 501
-
-
Manually annotated by BRENDA team
lipoyl domain of strain 232; strains J, Beaufor, 232, Sue, YZ, and C1735/2, single copy gene pdhC, organized in the pdhCD operon
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
potato
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
maize
Uniprot
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
Nov3r a lipoyl group-binding site inhibitor (related trifluoro-2-hydroxy-2-menthylpropionate compound) prevents pyruvate dehydrogenase kinase2 and GST-L2 (glutathione-S-transferase fused to the inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase) binding and dissect the effects of Nov3r binding at the lipoyl group binding site on PDHK2 binding of other ligands
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + enzyme N6-(dihydrolipoyl)lysine
CoA + enzyme N6-(S-acetyldihydrolipoyl)lysine
show the reaction diagram
-
reductive acetylation of the enzyme
-
-
?
dihydrolipoamide + acetyl-CoA
S-acetyldihydrolipoamide + CoA
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
dihydrolipoamide + acetyl-CoA
S-acetyldihydrolipoamide + CoA
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K+
-
association of the pyruvate dehydrogenase kinase2 and GST-L2 (glutathione-S-transferase fused to the inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase (E2)) dimers is enhanced by K+
phosphate
-
phosphate has a pronounced effect in increasing ligand interference with pyruvate dehydrogenase kinase2 and GST-L2 (glutathione-S-transferase fused to the inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase (E2))
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,2-diselenolane-3-valeric acid
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competitive with respect to dihydrolipoamide and non-competitive with respect to acetyl-CoA
CoA
-
competitive to acetyl-CoA, non-competitive to dihydrolipoamide
guanidine hydrochloride
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50% inhibition at 0.3 M, complete inhibition at 0.7-1 M
Iodine
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complete inactivation at 2.5 mol iodine per mol of subunit
lipoamidase
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complete loss of activity after 3 h
-
N-Acetylimidazole
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modifies tyrosyl residues and dissociates the enzyme into subunits
palmitoyl-CoA
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competitive to acetyl-CoA, non-competitive to dihydrolipoamide
S-acetyldihydrolipoamide
-
competitive to dihydrolipoamide, non-competitive to acetyl-CoA
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.013 - 0.1
acetyl-CoA
0.13 - 6.4
Dihydrolipoamide
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00417 - 2.4
acetyl-CoA
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.89
-
fusion protein with glutathione S-transferase
4.09
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after removal of glutathione S-transferase moiety
19.4
-
-
19.7
-
-
34.1
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purified 1-lip E2
47.1
-
catalytic domain
119
-
catalytic domain
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.8 - 8.5
-
-
8
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assay at
additional information
-
reactivity depending on buffer system
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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pyruvate dehydrogenase complex bound to inner mitochondrial membrane
Manually annotated by BRENDA team
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wild-type enzyme is soluble, whereas mutant enzyme with deletion of all three lipoyl domains aggregates
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
Pyrobaculum aerophilum (strain ATCC 51768 / IM2 / DSM 7523 / JCM 9630 / NBRC 100827)
Pyrobaculum aerophilum (strain ATCC 51768 / IM2 / DSM 7523 / JCM 9630 / NBRC 100827)
Pyrobaculum aerophilum (strain ATCC 51768 / IM2 / DSM 7523 / JCM 9630 / NBRC 100827)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
-
gel filtration in the presence of guanidinium hydrochloride
46930
calculation from nucleotide sequence
50080
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calculation from nucleotide sequence
51000
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gel filtration
52000 - 53000
SDS-PAGE
58000 - 59000
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SDS-PAGE
58890
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calculation from nucleotide sequence
59000
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SDS-PAGE
60100 - 64500
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sedimentation equilibrium, depending on buffer
63000
-
sedimentation equilibrium
64260
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calculation from nucleotide sequence
64910
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calculation from nucleotide sequence
65960
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calculation from nucleotide sequence
67000
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plastid precursor protein, SDS-PAGE
68000
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SDS-PAGE
73000 - 75000
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SDS-PAGE
78000
-
SDS-PAGE
81000
-
SDS-PAGE, gel filtration
127000
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fusion protein of 11000 Da fragment with beta-galactosidase, SDS-PAGE
150000
-
no component of pyruvate dehydrogenase complex, sucrose density gradient centrifugation
200000
500000
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above, gel filtration; gel filtration
530000
670000
864000
-
crystallization experiments
1000000
-
sedimentation equilibrium centrifugation
1470000
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sedimentation equilibrium experiments in 0.83 M acetic and and 0.005 M sodium chloride
1548000
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native enzyme
1560000
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fluorescence anisotropy decay experiments
1800000 - 1970000
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calculation from sedimentation and diffusion constants, meniscus depletion method
2700000
gel filtration
2800000
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calculation from subunit composition of pyruvate dehydrogenase complex
3100000
-
-
additional information
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
polymer
trimer
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3 * 82000, gel filtration, trimeric form occurs in solutions with 4 M guanidine hydrochloride
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
lipoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method
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pyruvate dehydrogenase complex
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E2-E3 subcomplex of pyruvate dehydrogenase, E2: EC 2.3.1.12, E3: EC 1.8.1.4, various crystallization conditions
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E3 dimer with E2 and a small binding domain E2BD, hanging drop vapor diffusion method
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crystal structure of pyruvate dehydrogenase kinase 2 bound to the inner lipoyl-bearing domain of dihydrolipoamide transacetylase is determined. Crystal structure reveals a pyruvate dehydrogenase kinase 2 dimer complexed with two inner lipoyl-bearing domains of dihydrolipoamide transacetylase. Comparison with apo-pyruvate dehydrogenase kinase 2 shows that dihydrolipoamide transacetylase binding causes rearrangements in PDHK2 structure that affect the dihydrolipoamidetransacetylase- and pyruvate dehydrogenase-binding sites
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 11
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isolated E. coli lipoyl domains stay soluble in this range
486166
6 - 8.4
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stable
486228
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
-
stable up to
78
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temperatures below result in less than 5% loss in enzyme activity
80
-
partly precipitation above
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
freezing and thawing does not cause loss of activity
-
sensitive to proteolysis
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-18°C, 0.05 mM potassium phosphate buffer, pH 7.0, 0.5 mM EDTA, more than 6 months
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liquid nitrogen, 50 mM potassium phosphate buffer pH 7.0, 0.5 mM EDTA, 0.05 mM phenylmethylsulfonylfluoride
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
catalytic domain mutants
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E1-E2 complex
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E2-E3 subcomplex of pyruvate dehydrogenase, i.e. dihydrolipoamide acetyltransferase and dehydrogenase
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from pyruvate dehydrogenase complex
fusion protein of 6 amino acids form beta-galactosidase, the apa-4 region and the catalytic domain of E2
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fusion protein of 6 amino acids form beta-galactosidase, the papa-4 region and the catalytic domain of E2
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fusion protein with glutathione S-transferase
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highly
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isolation of lipoyl domains
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peptides after limited proteolysis
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preparation of E2-X subcomplex: E2 i.e. EC 2.3.1.12, X i.e. component X of mammalian pyruvate dehydrogenase complex
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proteolytic fragments, isolation of lipoyl domain and catalytic domain
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purification from pyruvate dehydrogenase complex, composed of EC 1.2.4.1, EC 1.8.1.4, EC 2.3.1.12
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purification of pyruvate dehydrogenase complex, composed of EC 1.2.4.1, EC 1.8.1.4, EC 2.3.1.12
Hansenula miso
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purification of the multienzyme complex
purification of tryptic fragments
-
pyruvate dehydrogenase complex and tryptic fragments of E2
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pyruvate dehydrogenase complex, composed of EC 1.2.4.1, EC 1.8.1.4, EC 2.3.1.12
pyruvate dehydrogenase complex, composed of EC 1.2.4.1, EC 1.8.1.4. EC 2.3.1.12
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pyruvate dehydrogenase complex, stoichiometry E1:E2:E3 is 1.56:1:0.89
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recombinant 1-lip E2 and recombinant hybrid lipoyl domain by ammonium sulfate fractionation, ion exchange and hydrophobic interaction chromatography
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recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography
using a sephacryl S-400 HR column
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using Ni-nitrilotriacetate-agarose affinity chromatography
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wild-type and mutants with deletions of lipolyl domains
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a recombinant fragment hL2S (containing the second lipoyl domain (L2), second hinge region, E1-binding domain (S) and third hinge region of hE2, residues 128–330) is overexpressed in Escherichia coli
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catalytic domain, residues 173-427
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expressed in Escherichia coli
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expression in Escherichia coli
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expression of subgene for lipoyl domain in Escherichia coli
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expression of the enzymes' L2 domain as GST-fusion protein
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functional overexpression of the enzyme E2 with a single hybrid lipoyl domain per subunit, i.e. 1-lip E2, in strain JRG1342 and BL21(DE3), overexpression of the isolated hybrid lipoyl domain in strain JM 101
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gene pdhC, organized in the pdhCD operon, physical map, DNA and amino acid sequence determination and analysis, expression as His6-tagged enzyme in Escherichia coli
inner lipoyl domain (L2) of dihydrolipoyl acetyltransferase is expressed as a GST fusion protein
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knock-out mutants
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overexpression of truncated thrombin-cleavable enzyme in Escherichia coli strain BL21(DE3)
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the inner lipoyl-bearing domain of dihydrolipoamide transacetylase is expressed as a GST fusion protein in Escherichia coli
-
wild-type and deletion mutants, expression in Escherichia coli
-
wild-type and mutants
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K96A
-
mutation in potential lipoylation site, mutant protein entirely supports the assignment of Lys96 as the site of lipoylation
K96R
-
mutation in potential lipoylation site, mutant protein entirely supports the assignment of Lys96 as the site of lipoylation
G610C
-
500fold reduced enzymatic activity
N614D
-
9fold reduced enzymatic activity
S558A
-
200fold reduced enzymatic activity
K244Q
-
unlipoylatable domain
K136A
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
K153A
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
M131A
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
R135A
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
R135C
-
site-directed mutagenesis, additional alkylation of the mutant enzymes with methyl, ethyl, propyl and butyl groups, the modifications cause alterations in interaction of core unit, E1 and E3 in the enzyme complex compared to the wild-type complex, thermodynamics and kinetics in comparison to the wild-type enzyme, overview
R135K
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
R135L
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
R135M
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
R139A
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
R146A
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
R156A
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
S133A
-
site-directed mutagenesis, thermodynamics and kinetics in comparison to the wild-type enzyme
A174S
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: 69%
D164A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: 55%
D172A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: 66%
E162A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: 93%
E179A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: 28%
E182Q
-
binding of mutant L2 domain to pyruvate dehydrogenase phosphatase isozyme 1 is hindered
E183A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: 98%
F231A
-
ability to be post-translationally lipoylated remains
I176A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: not measurable
I229A
-
ability to be post-translationally lipoylated remains
I229A/F231A
-
ability to be post-translationally lipoylated remains, far-UV CD spectrum differs from wild-type enzyme
K173A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: not measurable
K276A
-
mutant shows negligible binding to human pyruvate dehydrogenase with 86fold higher KD compared to wild-type
L140A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: not measurable
L189A
-
ability to be post-translationally lipoylated remains
M194A
-
ability to be post-translationally lipoylated remains
M197A
-
ability to be post-translationally lipoylated remains
R196A
-
mutation in the inner lipoyl-bearing domain (L2), pyruvate dehydrogenase kinase 2 binding: 79%
R297A
-
mutant is found to have KD 6.8fold higher than that for the wild-type, indicating a possible involvement of this residue in the interaction with human pyruvate dehydrogenase, but not with the C-terminal residue of beta-subunit
V180S/E181L
-
binding of mutant L2 domain to pyruvate dehydrogenase phosphatase isozyme 1 is hindered
V188A
-
ability to be post-translationally lipoylated remains, far-UV CD spectrum differs from wild-type enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reconstitution of E1 with E2-E3 complex with a stoichiometry of E1:E2:E3 of 2:1:1
-
reconstitution of pyruvate dehydrogenase complex
reconstitution of pyruvate dehydrogenase complex with components from Escherichia coli and Salmonella typhimurium
-
reconstitution of pyruvate dehydrogenase complex, maximum activity is produced when transacetylase accommodates 12 pyruvate dehydrogenase dimers and 6 flavoprotein dimers
-
reconstitution of pyruvate dehydrogenase complex, optimum catalytic stoichiometry of E1:E2:E3 is 5.2:11.5:2
-
reconstitution of pyruvate dehydrogenase complex, stoichiometry pyruvate dehydrogenase:transacetylase:flavoprotein is 1:0.35:0.4
-
reconstitution of the active multienzyme complex with recombinant components
-
reconstitution with components from Escherichia coli and Azotobacter vinelandii, optimum catalytic stoichiometry of E1:E2:E3 is 1:1:0.5
-
treatment with dilute acetic acid solution results in dissociation into inactive subunits with MW 70000, removal of the acid results in restoration of enzymatic activity
-
treatment with guanidine hydrochloride and its subsequent removal results in little recovery of the core, but full recovery of X
-
treatment with guanidine hydrochloride and its subsequent removal results in refolding
-
treatment with guanidine hydrochloride results in dissociation into subunits, removal of guanidine hydrochloride results in refolding of the enzyme with 95% of the original activity
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
molecular biology
dihydrolipoamide acetyltransferase is shown to be a metabolic longevity factor and is required for calorie restriction-mediated life span extension
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