Information on EC 2.4.1.186 - glycogenin glucosyltransferase

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

EC NUMBER
COMMENTARY hide
2.4.1.186
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RECOMMENDED NAME
GeneOntology No.
glycogenin glucosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
UDP-alpha-D-glucose + glycogenin = UDP + alpha-D-glucosylglycogenin
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
glycogen biosynthesis
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glycogen biosynthesis II (from UDP-D-Glucose)
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SYSTEMATIC NAME
IUBMB Comments
UDP-alpha-D-glucose:glycogenin alpha-D-glucosyltransferase
The first reaction of this enzyme is to catalyse its own glucosylation, normally at Tyr-194 of the protein if this group is free. When Tyr-194 is replaced by Thr or Phe, the enzyme's Mn2+-dependent self-glucosylation activity is lost but its intermolecular transglucosylation ability remains [7]. It continues to glucosylate an existing glucosyl group until a length of about 5--13 residues has been formed. Further lengthening of the glycogen chain is then carried out by EC 2.4.1.11, glycogen (starch) synthase. The enzyme is not highly specific for the donor, using UDP-xylose in addition to UDP-glucose (although not glucosylating or xylosylating a xylosyl group so added). It can also use CDP-glucose and TDP-glucose, but not ADP-glucose or GDP-glucose. Similarly it is not highly specific for the acceptor, using water (i.e. hydrolysing UDP-glucose) among others. Various forms of the enzyme exist, and different forms predominate in different organs. Thus primate liver contains glycogenin-2, of molecular mass 66 kDa, whereas the more widespread form is glycogenin-1, with a molecular mass of 38 kDa.
CAS REGISTRY NUMBER
COMMENTARY hide
117590-73-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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-
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Manually annotated by BRENDA team
Coturnix sp.
Quail
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
hen
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
Hampshire crossbred animals with and without presence of th RN- allele. The total muscle glucose content 0.5 h post-mortem is 77% higher in RN- carriers compared with wild type animals. A greater transcription of glycogenin is found in RN- carriers. The glycogenin m-RNA is more abundant in RN- carriers compared with wild type animals.
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
CDP-glucose + glycogenin
CDP + glucosylated glycogenin
show the reaction diagram
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recombinant enzyme expressed in E. coli, 71% activity compared to UDP-glucose
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?
CDP-glucose + p-nitrophenyl-alpha-maltoside
CDP + glucosylated p-nitrophenyl-alpha-maltoside
show the reaction diagram
-
recombinant enzyme expressed in E. coli
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-
?
TDP-glucose + glycogenin
TDP + glucosylated glycogenin
show the reaction diagram
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recombinant enzyme expressed in E. coli, 33% activity compared to UDP-glucose
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?
TDP-glucose + p-nitrophenyl-alpha-maltoside
TDP + glucosylated p-nitrophenyl-alpha-maltoside
show the reaction diagram
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recombinant enzyme expressed in E. coli
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-
?
UDP-alpha-D-glucose + glycogenin
UDP + alpha-D-glucosylglycogenin
show the reaction diagram
UDP-galactose + glycogenin
UDP + galactosylated glycogenin
show the reaction diagram
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autoglycosylation reaction
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?
UDP-glucose + glycogenin
UDP + glucosylated glycogenin
show the reaction diagram
UDP-glucose + glycogenin
UDP + glucosylglycogenin
show the reaction diagram
UDP-glucose + maltose
UDP + maltotriose
show the reaction diagram
trans-glucosylation. 93% of the transferred glucose molecules appears in maltotriose, 6% are attached to glycogenin, and 1% is liberated as free glucose
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-
?
UDP-glucose + N-(maltosyl-alpha-1,4-(1-deoxyglucitol))-peptide
UDP + glucosylated N-(maltosyl-alpha-1,4-(1-deoxyglucitol))-peptide
show the reaction diagram
UDP-glucose + n-dodecyl-beta-D-maltoside
UDP + n-dodecyl-beta-D-maltotriose
show the reaction diagram
UDP-glucose + n-octyl-alpha-D-maltoside
?
show the reaction diagram
UDP-glucose + n-octyl-beta-D-maltoside
?
show the reaction diagram
UDP-glucose + n-tetradecyl-beta-D-maltoside
?
show the reaction diagram
UDP-xylose + glycogenin
UDP + xylosylated glycogenin
show the reaction diagram
UDP-xylose + n-dodecyl-beta-D-maltoside
?
show the reaction diagram
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transglucosylation reaction
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-
?
additional information
?
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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
UDP-alpha-D-glucose + glycogenin
UDP + alpha-D-glucosylglycogenin
show the reaction diagram
UDP-glucose + glycogenin
UDP + glucosylated glycogenin
show the reaction diagram
UDP-glucose + glycogenin
UDP + glucosylglycogenin
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ADP
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skeletal muscle enzyme, allosteric inhibition of autoglucosylation
CDP
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renal enzyme, 90% inhibition at 0.025 mM
CDP-choline
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renal enzyme, 75% inhibition at 0.1 mM
CDP-glucose
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maltose
phosphate
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slight inhibition at 100 mM
TDP-glucose
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UDP-xylose
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competitive inhibitor to glucosylation of glycogenin by UDP-glucose
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
Coturnix sp.
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enzyme is phosphorylated in embryonic muscle
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1
n-dodecyl-beta-D-maltoside
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3
p-nitrophenyl-alpha-maltoside
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0.002 - 0.00441
UDP-glucose
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0053 - 0.0183
UDP-glucose
additional information
additional information
Oryctolagus cuniculus
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the catalytic effectiveness of glycogenin falls off dramatically as the average glucosyl chain length increases
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
Coturnix sp.
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Manually annotated by BRENDA team
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the samples for gene expression analyses are taken 0.5 h after slaughter
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
31000
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SDS-PAGE
32000
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kidney enzyme
34000
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non-glycosylated truncated protein, gel filtration of a 0.0005 mM solution
37280
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amino acid sequence determination
40000
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recombinant His-tagged unglucosylated glycogenin-1, gel filtration
41000
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recombinant His-tagged autoglucosylated glycogenin-1, gel filtration
47000
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a glycogenin species of average molecular weight 47000 Da is isolated from proteoglycogen isoamylolyzed for 4.5 h, SDS-PAGE
58000
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truncation mutant enzymes DELTA270-332/D159S and DELTA270-332/D162S, gel filtration
59000
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non-glycosylated truncated protein, gel filtration of a 0.02 mM solution
103000
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wild-type enzyme in glycosylated and nonglycosylated form, full-length mutant enzymes D162S and D159S, gel filtration
124000
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gel filtration, heterodimeric glycogen synthase complex
200000
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gel filtration, active proteoglycogen
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 31000, SDS-PAGE
monomer
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1 * 34000, non-glycosylated truncated protein, 0.0005 mM enzyme solution; 1 * 38000, native enzyme, 50-70% of the activity of the dimer
tetramer
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mutant enzymes D159N, D162N and DELTA270-332/D162N exist as both tetrameric and dimeric species
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant hGYG1 in complex with Mn2+ and UDP-alpha-glucose, 10 mg/ml hGYG1 with various ligands by sitting drop vapor diffusion at 20C, X-ray diffraction structure determination and analysis at 2.6 A, molecular replacement
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purified recombinant glycogenin-1 truncation mutant DELTA-270, hanging drop vapor diffusion method, the protein in 12% w/v, PEG monomethyl ester 5000, 0.1 M MES, pH 6.5, and 0.2 M ammonium sulfate, is mixed with 1 M ammonium sulfate and 0.1 M sodium phosphate, pH 6.7, 4C, crystals are soaked in mother liquor containing 1 mM MnSO4 and 1 mM UDP or UDP-glucose, X-ray diffraction structure determination and analysis
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the DELTA270 enzyme crystallizes at 7.5 mg/ml from solutions containing 1 mM UDP-glucose, 1 mM MnCl2, 100 mM sodium acetate, pH 4.54.7, and 1013% (w/v) PEG 4000 in the space group P64, with cell dimensions a = b = 75.0, c = 233.4, gamma = 120 and a dimer in the asymmetric unit. All crystals are grown using hanging drop vapor diffusion methods at 23C
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
alkali-stable
concentrated to 8 mg/ml, and 100 microl aliquots are flash frozen in liquid nitrogen and stored at -80C. Glycogenin stored in this manner is stable for at least 2 years.
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loss of activity on Q-Sepharose during ion exchange chromatography
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, amylolyzed M-glycogenin preparation, at least 1 month without appreciable loss of activity
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4C, amylolyzed M-glycogenin preparation, at least 1 week without appreciable loss of activity
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-Sepharose column chromatography and Q Sepharose column chromatography
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nickel-NTA column and Q-Sepharose column
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recombinant His-tagged wild-type and mutant enzymes from cell-free expression by nickel affinity chromatography
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chrmatography
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recombinant proteins
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recombinant wild-type gycogenin-1, its truncated mutant DELTA-270, and of glycogenin-1 point mutants from Escherichia coli strain CGSC 4997
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separation from glycogen synthase, EC 2.4.1.11, by LiBr
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separation from proteoglycogen
wild-type and mutant enzymes
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cell-free expression of His-tagged wild-type and mutant enzymes
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expressed in C2C12 cells
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expressed in Escherichia coli strain Rosetta (DE3)
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expression in COS cells; expression of mutant Y194F in Escherichia coli
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expression in Escherichia coli as a GST-tagged protein
expression of glucose-free apo-glycogenin in an Escherichia coli mutant lacking UDP-glucose, enzyme is active towards itself and other substrates
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expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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expression of mutant Y194F in Escherichia coli; functional expression of the wild-type enzyme in Escherichia coli
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expression of wild-type and mutant enzymes in Saccharomyces cerevisiae
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expression of wild-type gycogenin-1, its truncated mutant DELTA-270, and of glycogenin-1 point mutants in Escherichia coli strain CGSC 4997
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expresssion of recombinant protein in the CGSC Escherichia coli cell line 4997, which lacks UDP-glucose pyrophosphorylase activity
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isolation of cDNA
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preparation of a truncated already glucosylated His-tag fusion protein in Escherichia coli ER2566, expression of the truncated non-glucosylated His-tag fusion protein in Escherichia coli CGSC 4997
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
T83A
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site-directed mutagenesis, the mutant shows no incorporation of glucose, no autoglycosylation
T83C
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site-directed mutagenesis, the mutant shows no incorporation of glucose, no autoglycosylation
T83F
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site-directed mutagenesis, the mutant shows no incorporation of glucose, no autoglycosylation
T83S
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site-directed mutagenesis, the mutant is catalytically active
T83V
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site-directed mutagenesis, the mutant shows no incorporation of glucose, no autoglycosylation
T83Y
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site-directed mutagenesis, the mutant shows no incorporation of glucose, no autoglycosylation
Y195F
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site-directed mutagenesis, glycogenin-1 with the Thr83Met substitution is unable to form the glucose-O-tyrosine linkage at tyrosine 195 unless co-expressed with the catalytically active Tyr195Phe glycogenin-1
DELTA306-664
7fold increase in self-glucosylation
DELTA306-664/Y196F
no self-glucosylation activity
DELTA306-664/Y196F/Y198F
expression results in no accumulation of glycogen
DELTA360-664
11.8fold increase in self-glucosylation
DELTA360-664/Y196F
expression results in reduced glycogen accumulation to 30% of the wild-type enzyme, very low self-glucosylation activity
DELTA360-664/Y196F/Y198F
no self-glucosylation activity
D159N
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exists as both tetrameric and dimeric species, compared to wild-type enzyme which exists to more than 95% as dimer, self-glucosylation activities below the limit of detection of the assay. Ability to catalyze the transglucosylation of maltose is reduced by 260fold, hydrolysis of UDP-glucose is reduced 12fold
D159S
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stable enzyme, self-glucosylation activities below the limit of detection of the assay. Transglucosylation activity of the mutant enzyme is reduced to undetectable levels, activity for the hydrolysis of UDP-glucose is reduced 14fold
D162N
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exists as both tetrameric and dimeric species, compared to wild-type enzyme which exists to more than 95% as dimer, self-glucosylation activities below the limit of detection of the assay, undetectable activity for the transglucosylation of maltose and the hydrolysis of UDP-glucose to free glucose
D162S
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stable enzyme, self-glucosylation activities below the limit of detection of the assay. 30fold less active for the trans-glucosylation of maltose and 340fold less active for the hydrolysis of UDP-glucose
DELTA270-332
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mutant enzyme is fully active, specific activity for self- or transglucosylation is indistinguishable from the full-length enzyme
DELTA270-332/D159S
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inactive mutant enzyme
DELTA270-332/D162N
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exists as both tetrameric and dimeric species, compared to wild-type enzyme which exists to more than 95% as dimer
DELTA270-332/D162S
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18fold less active for the transglucosylation of maltose and 190fold less active for the hydrolysis of UDP-glucose than wild-type enzyme, activity for the hydrolysis of UDP-glucose is reduced 4fold
T82M
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inactive mutant, the mutation is equivalent to T83M according to previous authors amino acid numbering, it causes glycogenosis showing the loss of Thr82 hydrogen bond to Asp162, the residue involved in the activation step of the glucose transfer reaction mechanism. Autoglucosylation, maltoside transglucosylation and UDP-glucose hydrolyzing activities are abolished
T83S
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site-directed mutagenesis, the mutant is catalytically active
T83V
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site-directed mutagenesis, inactive mutant
Y194F
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exchange of glucose attachment site, no autoglucosylation activity
Y194X
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mutation at Y194 leads to a protein unable to attach glucose to itself
Y194T
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exchange of glucose attachment site, no autoglucosylation activity, mutant glycosylates other substrates but with less activity compared to the wild-type
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
synthesis
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the COOH-terminal fragment of glycogenin can be used as a effective high affinity reagent for the purification of glycogen synthase from skeletal muscle and liver