Information on EC 2.5.1.61 - hydroxymethylbilane synthase and Organism(s) Homo sapiens and UniProt Accession P08397

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Homo sapiens
UNIPROT: P08397


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


The taxonomic range for the selected organisms is: Homo sapiens

EC NUMBER
COMMENTARY hide
2.5.1.61
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RECOMMENDED NAME
GeneOntology No.
hydroxymethylbilane synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
4 porphobilinogen + H2O = hydroxymethylbilane + 4 NH3
show the reaction diagram
catalytic cycle, cofactor assembly and tetrapyrrole chain polymerization mechanism, C261 is involved, residues D99 and R167 are essential for activity, overview
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
tetrapyrrole biosynthesis I (from glutamate)
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tetrapyrrole biosynthesis II (from glycine)
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heme metabolism
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Porphyrin and chlorophyll metabolism
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Metabolic pathways
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Biosynthesis of secondary metabolites
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
porphobilinogen:(4-[2-carboxyethyl]-3-[carboxymethyl]pyrrol-2-yl)methyltransferase (hydrolysing)
The enzyme works by stepwise addition of pyrrolylmethyl groups until a hexapyrrole is present at the active centre. The terminal tetrapyrrole is then hydrolysed to yield the product, leaving a cysteine-bound dipyrrole on which assembly continues. In the presence of a second enzyme, EC 4.2.1.75 uroporphyrinogen-III synthase, which is often called cosynthase, the product is cyclized to form uroporphyrinogen-III. If EC 4.2.1.75 is absent, the hydroxymethylbilane cyclizes spontaneously to form uroporphyrinogen I.
CAS REGISTRY NUMBER
COMMENTARY hide
9036-47-9
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9074-91-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4 porphobilinogen + H2O
hydroxymethylbilane + 4 NH3
show the reaction diagram
porphobilinogen + H2O
hydroxylmethylbilane + NH3
show the reaction diagram
-
-
-
-
?
porphobilinogen + H2O
hydroxymethylbilane + NH3
show the reaction diagram
porphobilinogen + H2O
1-hydroxymethylbilane + 4 NH3
show the reaction diagram
porphobilinogen + H2O
hydroxylmethylbilane + NH3
show the reaction diagram
porphobilinogen + H2O
hydroxymethylbilane + 4 NH3
show the reaction diagram
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polymerization of 4 pyrrole molecules to a linear tetrapyrrole
i.e. pre-uroporphyrinogen, highly unstable compound
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?
porphobilinogen + H2O
hydroxymethylbilane + NH3
show the reaction diagram
porphobilinogen + H2O
uroporphyrinogen III
show the reaction diagram
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
4 porphobilinogen + H2O
hydroxymethylbilane + 4 NH3
show the reaction diagram
porphobilinogen + H2O
hydroxymethylbilane + NH3
show the reaction diagram
P08397
the enzyme is important in heme biosynthesis, a nonsense mutation in the porphobilinogen deaminase gene causes chester porphyria, existence of dual porphyrias with two enzymes of heme biosynthesis being deficient simultaneously, overview
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?
porphobilinogen + H2O
1-hydroxymethylbilane + 4 NH3
show the reaction diagram
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third reaction step in heme biosynthesis pathway
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-
?
porphobilinogen + H2O
hydroxymethylbilane + NH3
show the reaction diagram
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
dipyrromethane
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dipyrromethane
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cofactor assembly mechanism, R149 and R173 are important, cofactor is produced from the apo-enzyme with pre-uroporphyrinogen
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-methylopsopyrroledicarboxylic acid
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Cd2+
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12 mM, complete inhibition
Coproporphyrinogen
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Cu2+
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1 mM CuSO4, 87% inhibition
Fe2+
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1 mM FeSO4, 58% inhibition
Fe3+
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1 mM FeCl3, 62% inhibition
glycerol
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15% inhibits 40% of enzyme activity
Hg2+
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0.0004 mM HgCl2, 80% inhibition
isoporphobilinogen
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N-ethylmaleimide
p-chloromercuribenzoate
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0.002 mM, 74% inhibition
p-hydroxymercuribenzoate
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PbNO3
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0.005 mM, 35% inhibition
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protoporphyrinogen
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inhibits both erythroid and lymphoblast forms of the enzyme
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00342 - 1.579
porphobilinogen
0.051 - 0.077
Hydroxymethylbilane
0.0042 - 0.13
porphobilinogen
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.25
porphobilinogen
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.002
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R167Q mutant, pH 8.0
0.023
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wild type, pH 8.0
0.000039
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purified recombinant GST-tagged mutant enzyme
0.00117
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0.0128
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40 kDa enzyme form
0.0183
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42 kDa enzyme form
0.022
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purified recombinant GST-tagged wild-type enzyme
0.038
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A form
0.039
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B form
0.04
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0.0402
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additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.4
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phosphate buffer
7.8
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mutant enzyme T59I
8
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mutant enzyme V215M
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9
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pH 5.0: unstable below, pH 9.0 stable above
6 - 9
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pH 6.0: no activity below, pH 9.0: 50% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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assay at
37
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assay at
45
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activity twice that at 37C
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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peripheral blood
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
Homo sapiens;
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42000
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wild type and mutant recombinant enzymes except truncated enyzme p.Ala226ProfsX28
44000
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53000
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GST-tagged recombinant p.Ala226ProfsX28, removing the GST tag produces a strongly degraded enzyme
68000
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glutathione S-transferase (GST)-tagged wild type and recombinant enzymes except p.Ala226ProfsX28
25000
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gel filtration
37000
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1 * 37000, SDS-PAGE, A and B form
39500
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1 * 39500, SDS-PAGE, B2 and B3
40000
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1 * 40000, SDS-PAGE
41000
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1 * 41000, SDS-PAGE
41200
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1 * 41200, SDS-PAGE, erythrocyte enzyme
44000
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gel filtration
68000
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x * 68000, GST-tagged recombinant enzyme, SDS-PAGE, x * 42000, detagged recombinant enzyme, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 44000, about, recombinant detagged wild-type and mutant enzymes, SDS-PAGE
?
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x * 68000, GST-tagged recombinant enzyme, SDS-PAGE, x * 42000, detagged recombinant enzyme, SDS-PAGE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
vapor diffusion method, hanging drops with 20 mM Tris-HCl buffer, pH 8.2, containing 5 mM dithiothreitol and reservoir solution consisting of 0.6 M ammonium sulfate, 1.2 M lithium sulfate, 5% ethylene glycol, 50 mM sodium citrate, pH 5.6, and 50 mM dithiothreitol, diffraction data are collected at -173C in 30% glycerol cryoprotected protein crystals
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
65
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at pH 8.2, wild type with 30% activity loss after 240 min, mutant Q204K with a half-life of 100 min has about one third of the stability of the wild type
56
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2h, no loss of activity
60
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2 h, 10% loss of activity
65
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15 min, no loss of activity
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
DTT stabilizes
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phosphate stabilizes
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
photooxidation of the enzyme in the presence of methylene blue, 0.03%, and roes bengal, 0.03%, over a 5 min period, inhibits 90% and 60% of the enzyme activity, respectively
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5883
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15C, purified enzyme rapidely loses its activity
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-20C, forms A to E, stable for up to 18 months in 10 mM potassium phosphate buffer, pH 8.0, containing 0.2 mM dithioerythritol
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4C, purified enzyme stable for more than a month
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erythrocytes lysates stored in 1 mM potassium phosphate, pH 7.6, containing 0.05% Trition X-100, 1 mM dithiothreitol and 1 mM MgCl2, no or little loss of activity after 1 year
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purified enzyme loses its activity when it is frozen
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
blood samples are washed in phosphate buffered saline, liver rinsed in phosphate buffered saline
cell harvesting by centrifugation, washed, resuspended in NaCl-phosphate buffer containing of 140 mN NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3 with protease inhibitor cocktail, and 0.5% Triton X-100, lysis by shaking with lysozyme on ice, sonication, centrifugation, supernatant loaded onto glutathione sepharose 4B column, washed with 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40+, pH 8.2, proteins eluted in 50 mM Tris-HCl, pH 8.3 buffer with 20 mM glutathione, thrombin digestion with thrombin at 20 U/mg protein at 20C overnight, addition of glycerol to a concentration of 20%
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cells centrifuged, washed with 0.9 M NaCl, re-centrifuged, washed with 20 mM Tris-HCl buffer, pH 8.2, with 5 mM dithiothreitol, and 200 microM PMSF, sonication, heating to 60C under N2 gas, cooled to 4C, supernatant applied to a Pharmacia 50 K/30 column packed with DEAE-Sephacel anion-exchange resin, equilibrated with 50 mM Tris-HCl buffer, pH 8.2 containing 5 mM dithiothreitol, and 100 microM PMSF, elution with 0-70 mM KCl gradient, active fractions are pooled and concentrated by ultrafiltration with a PM-10 membrane, further concentrated with a Centricon YM-10 centrifugal filter and gel-filtered on a Hiload 16/60 Superdex G-75 column, equilibrated with 100 mM Tris-HCl buffer, pH 8.2, containing 5 mM dithiothreitol and 100 microM PMSF, concentrated with a Centricon YM-10, buffer-exchanged into 20 mM Tris-HCl buffer, pH 8.2, containing 5 mM dithiothreitol with a Pharmacia PD10 column
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recombinant GST-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) pLysS by glutathione affinity chromatgraphy, tag cleavage by thrombin, and ultrafiltration
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2 forms
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3 isoenzymes
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5 forms, A: native enzyme, B-E: isomeres corresponding to the enzyme-substrate intermediates
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glutathione Sepharose 4B column chromatography
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partial
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recombinant GST-tagged isoallelic forms K210 and E210 mutants from Escherichia coli to homogeneity
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recombinant GST-tagged wild-type and mutant enzymes from Escherichia coli BL21 (DE3) by glutathione affinity chromatography, the GST-tag is cleaved off
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Escherichia coli BL21(DE3) pLysS with pUHD2 plasmid
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expression in Escherichia coli BL21(DE3) with pGEX-4T-1 expression vector
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localization of the porphobilinogen deaminase gene on chromosome 11q23.3
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pTRE-EalAAT-hPBGD-WPRE plasmid from pTRE2 vector expressed in mice hepatocytes
recombinant expression of GST-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) pLysS
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cloning and sequencing of naturally occurring mutants G11R and R173Q
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expressed in Escherichia coli BL21 cells
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expression of wild-type and mutant enzymes in SH-SY5Y neuroblastoma cells, expression analysis
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expression of wild-type and mutants in Escherichia coli strain BL21
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PBGD gene, DNA and amino acid sequence determination and analysis, expression of GST-tagged wild-type and mutant enzymes in Escherichia coli BL21 (DE3)
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the enzyme is expressed into a housekeeping or an erythroid form as a result of differential promoter usage and splicing, three pairs of isoallelic forms, expression of two of the isoallelic forms, K210 and E210, with mutations involved in acute intermittent porphyria, AIP, in Escherichia coli as GST fusion proteins
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
transfer of therapeutic plasmid with human enzyme into hepatocytes
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G24S
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c.70G>A (p.Gly24Ser)
H300L
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c.899_900delinsTGCCTGCATCTG (p.His300LeuFsX10)
K132N
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site-directed mutagenesis, the mutant shows no conformational or kinetic defect, no loss in relative activity (97% of wild-type activity) at standard conditions nor change in Vmax and Km. The mutation is not associated to acute intermittent porphyria, AIP
N322K
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c.965_966insA (p.Asn322LysfsX36)
Q204K
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c.610C>A, exon 10, missense mutation, 46% wild type activity, 50 mM Tris-HCl, pH 8.2, 0.1% bovine serum albumin, 0.1% Triton, pH 8.2, 37C, 1 h in the dark
R116W
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site-directed mutagenesis, the mutant shows 0.5% of wild-type activity and defects in conformational stability. The mutation is associated to acute intermittent porphyria, AIP
R149X
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identification of a nonsense mutation in the PBGD gene on chromosome 11q23.3, which harbors the mutations causing acute intermittent porphyria, as the underlying genetic defect in Chester porphyria, phenotype, overview
R167Q
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8.5% activity, reduced pH optimum from 8.0 to 6.0, accumulates long-lived intermediate complexes
R167W
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site-directed mutagenesis, the mutant shows 4.2% of wild-type activity and defects in enzyme kinetics associated with a very high Km and decreased Vmax. The mutation is associated to acute intermittent porphyria, AIP
R173Q
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c.518G>A, exon 10, missense mutation, 0.15% wild type activity, 50 mM Tris-HCl, pH 8.2, 0.1% bovine serum albumin, 0.1% Triton, pH 8.2, 37C, 1 h in the dark
R173Q/Q204K
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R173Q is more severe with a resulting enzyme activity of nearly zero, the Q204K increases the negative effect, particularly on the protein stability, 50 mM Tris-HCl, pH 8.2, 0.1% bovine serum albumin, 0.1% Triton, pH 8.2, 37C, 1 h in the dark
R173W
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site-directed mutagenesis, the mutant shows 0.6% of wild-type activity and defects in conformational stability and in enzyme kinetics. The mutation is associated to acute intermittent porphyria, AIP
R26H
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c.77G>A, exon 3, 0.2% wild type activity, 50 mM Tris-HCl, pH 8.2, 0.1% bovine serum albumin, 0.1% Triton, pH 8.2, 37C, 1 h in the dark
R325A
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c.972_973insG (p.Arg325AlafsX33)
R32P
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c.95G>C (p.Arg32Pro)
R73Q/Q204K
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a complex monoallelic mutation c.[518G>A; c.610C>A] (p.[Arg173Gln;p.Gln204Lys])
T59I
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c.176C>T (p.Thr59Ille)
V215E
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site-directed mutagenesis, the mutant shows 30% of wild-type activity and lower conformational stability and probably a perturbed elongation process, also 70% loss in both activity and Vmax. The mutation is associated to acute intermittent porphyria, AIP
887insA
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site-directed mutagenesis, the mutant shows reduced expression and subcellular distribution compared to the wild-type enzyme
D99G
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site-directed mutagenesis, inactive holo-enzyme mutant existing as a complex with 2 substrate molecules covalently bound to the dipyrromethane cofactor
G748A
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site-directed mutagenesis, the mutant shows reduced expression and subcellular distribution compared to the wild-type enzyme
G748C
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site-directed mutagenesis, the mutant shows reduced expression and subcellular distribution compared to the wild-type enzyme
R149Q
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site-directed mutagenesis, inactive apo-enzyme which is unable to assemble with the dipyrromethane cofactor, the mutant enzyme is unstable and heat-labile
R167Q
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site-directed mutagenesis, mutation of active site residue, highly reduced activity compared to the wild-type enzyme, formation of stable enzyme-intermediate complexes
R173Q
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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme, the major part of the enzyme exists as apo-enzyme without assembled dipyrromethane cofactor
T59I
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80.6% residual activity compared to the wild type enzyme
V215M
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19.4% residual activity compared to the wild type enzyme
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
medicine
enzyme over-expression in hepatocytes reduces precursor accumulation, liver-directed gene therapy might offer an alternative to liver transplantation
medicine
pharmacology
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safety, pharmacokinetics and pharmacodynamics of recombinant human porphobilinogen deaminase P 9808, administered to healthy subjects and asymptomatic porphobilinogen deaminase-deficient subjects with high concentrations of porphobilinogen, the substrate of porphobilinogen deaminase for investigation and establishing of an alternative therapy of acute intermittent porphyria, AIP, overview