Application | Comment | Organism |
---|---|---|
diagnostics | molecular analysis of the hydroxymethylbilane synthase confirms risk for acute intermittent porphyria | Homo sapiens |
Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli BL21(DE3) with pGEX-4T-1 expression vector | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
A226P | c.675delA, exon 12, 0.05% wild type activity, small deletion leads to stop codon after 28 completely different amino acids compared to wild type, 50 mM Tris-HCl, pH 8.2, 0.1% bovine serum albumin, 0.1% Triton, pH 8.2, 37°C, 1 h in the dark | Homo sapiens |
E250D | c.750A>T, exon 12, missense mutation, 0.5% wild type activity, 50 mM Tris-HCl, pH 8.2, 0.1% bovine serum albumin, 0.1% Triton, pH 8.2, 37°C, 1 h in the dark | Homo sapiens |
additional information | previously known mutations that are identified by molecular analysis of the hydroxymethylbilane synthase gene are c.76C>T (R26Cys), c.77G>A (R26H), c.518G>A (R173Q), c.771 + 1G>T (causing donor splice site defect, deletion of exon 12), novel mutations are c.610C>A (Q204K), c.675delA (A226P with stop codon 28), c.750A>T (E250D), one patient shows double mutation of R173Q and Q204K | Homo sapiens |
Q204K | 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, 37°C, 1 h in the dark | Homo sapiens |
R173Q | 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, 37°C, 1 h in the dark | Homo sapiens |
R173Q/Q204K | 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, 37°C, 1 h in the dark | Homo sapiens |
R26C | c.76C>T, exon 3, 0.3% wild type activity, 50 mM Tris-HCl, pH 8.2, 0.1% bovine serum albumin, 0.1% Triton, pH 8.2, 37°C, 1 h in the dark | Homo sapiens |
R26H | 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, 37°C, 1 h in the dark | Homo sapiens |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00342 | - |
porphobilinogen | mutant Q204K, Vmax is reduced 3fold to 0.66 nmol/min compared to the wild type, 50 mM Tris-HCl, pH 8.2 | Homo sapiens | |
0.00445 | - |
porphobilinogen | wild type, Vmax = 2.14 nmol/min, 50 mM Tris-HCl, pH 8.2 | Homo sapiens |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
42000 | - |
wild type and mutant recombinant enzymes except truncated enzyme p.Ala226ProfsX28 | Homo sapiens |
53000 | - |
GST-tagged recombinant p.Ala226ProfsX28, removing the GST tag produces a strongly degraded enzyme | Homo sapiens |
68000 | - |
glutathione S-transferase (GST)-tagged wild type and recombinant enzymes except p.Ala226ProfsX28 | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
4 porphobilinogen + H2O | Homo sapiens | - |
hydroxymethylbilane + 4 NH3 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | P08397 | - |
- |
Purification (Comment) | Organism |
---|---|
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 20°C overnight, addition of glycerol to a concentration of 20% | Homo sapiens |
Source Tissue | Comment | Organism | Textmining |
---|---|---|---|
leukocyte | peripheral blood | Homo sapiens | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4 porphobilinogen + H2O | - |
Homo sapiens | hydroxymethylbilane + 4 NH3 | - |
? |
Synonyms | Comment | Organism |
---|---|---|
HMBS | - |
Homo sapiens |
hydroxymethylbilane synthase | - |
Homo sapiens |
porphobilinogen deaminase | - |
Homo sapiens |
uroporphyrinogen I synthase | - |
Homo sapiens |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
65 | - |
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 | Homo sapiens |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.2 | - |
wild type and mutant Q204K, measured between pH 7 and 9 | Homo sapiens |
General Information | Comment | Organism |
---|---|---|
malfunction | mutations cause acute intermittent porphyria | Homo sapiens |
metabolism | third enzyme in heme biosynthesis | Homo sapiens |