Literature summary for 2.5.1.61 extracted from

Ulbrichova, D.; Hrdinka, M.; Saudek, V.; Martasek, P.
Acute intermittent porphyria--impact of mutations found in the hydroxymethylbilane synthase gene on biochemical and enzymatic protein properties (2009), FEBS J., 276, 2106-2115.

Application

Application	Comment	Organism
diagnostics	molecular analysis of the hydroxymethylbilane synthase confirms risk for acute intermittent porphyria	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli BL21(DE3) with pGEX-4T-1 expression vector	Homo sapiens

Protein Variants

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 [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 [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/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
4 porphobilinogen + H2O	Homo sapiens	-	hydroxymethylbilane + 4 NH3	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P08397	-	-

Purification (Commentary)

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

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

Source Tissue	Comment	Organism	Textmining
leukocyte	peripheral blood	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
4 porphobilinogen + H2O	-	Homo sapiens	hydroxymethylbilane + 4 NH3	-	?

Synonyms

Synonyms	Comment	Organism
HMBS	-	Homo sapiens
hydroxymethylbilane synthase	-	Homo sapiens
porphobilinogen deaminase	-	Homo sapiens
uroporphyrinogen I synthase	-	Homo sapiens

Temperature Stability [°C]

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

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

General Information	Comment	Organism
malfunction	mutations cause acute intermittent porphyria	Homo sapiens
metabolism	third enzyme in heme biosynthesis	Homo sapiens