Information on EC 4.3.2.2 - adenylosuccinate lyase

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

EC NUMBER
COMMENTARY hide
4.3.2.2
-
RECOMMENDED NAME
GeneOntology No.
adenylosuccinate lyase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
(S)-2-[5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido]succinate = fumarate + 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide
show the reaction diagram
N6-(1,2-dicarboxyethyl)AMP = fumarate + AMP
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
beta-elimination
elimination
-
-
C-N bond cleavage
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
adenosine ribonucleotides de novo biosynthesis
-
-
Alanine, aspartate and glutamate metabolism
-
-
Biosynthesis of antibiotics
-
-
Biosynthesis of secondary metabolites
-
-
inosine-5'-phosphate biosynthesis I
-
-
inosine-5'-phosphate biosynthesis II
-
-
inosine-5'-phosphate biosynthesis III
-
-
Metabolic pathways
-
-
purine metabolism
-
-
Purine metabolism
-
-
SYSTEMATIC NAME
IUBMB Comments
N6-(1,2-dicarboxyethyl)AMP AMP-lyase (fumarate-forming)
Also acts on 1-(5-phosphoribosyl)-4-(N-succinocarboxamide)-5-aminoimidazole.
CAS REGISTRY NUMBER
COMMENTARY hide
9027-81-0
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
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
(Nomega-L-arginino)succinate
fumarate + L-arginine
show the reaction diagram
-
-
-
-
?
(S)-2-[5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido]succinate
fumarate + 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide
show the reaction diagram
-
-
-
-
?
(S)-2-[5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido]succinate
fumarate + 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido
show the reaction diagram
1-(5-phosphoribosyl)-4-(N-succinocarboxamide)-5-aminoimidazole
5'-phosphoribosyl-5-amino-4-imidazolecarboxamide
show the reaction diagram
1-(5-phosphoribosyl)-4-(N-succinocarboxamide)-5-aminoimidazole
5'-phosphoribosyl-5-amino-4-imidazolecarboxamide + fumarate
show the reaction diagram
-
-
-
-
?
2',3'-dideoxyadenylosuccinate
dideoxyadenosine 5'-monophosphate + ?
show the reaction diagram
-
at 1.44% of the activity with the natural substrate
-
?
2'-deoxy-succino-AMP
2'-deoxy-AMP + fumarate
show the reaction diagram
4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide
5-aminoimidazole-4-carboxamide ribonucleotide + fumarate
show the reaction diagram
5-aminoimidazole-(N-succinylocarboxamide) ribotide
5-aminoimidazole-4-carboxamide ribotide + fumarate
show the reaction diagram
5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide
5-aminoimidazole-4-carboxamide ribonucleotide + fumarate
show the reaction diagram
8-aza-succino-AMP
8-aza-AMP + fumarate
show the reaction diagram
-
-
-
?
adenylosuccinate
adenosine monophosphate + fumarate
show the reaction diagram
adenylosuccinate
AMP + fumarate
show the reaction diagram
adenylosuccinate 2',3'-acyclic dialcohol
adenylic 2',3'-acyclic dialcohol + fumarate
show the reaction diagram
-
-
-
-
?
adenylosuccinate 2',3'-acyclic dialdehyde
?
show the reaction diagram
-
-
-
?
arabinosyl-succino-AMP
arabinosyl-AMP + ?
show the reaction diagram
fumarate + AMP
N6-(1,2-dicarboxyethyl)AMP
show the reaction diagram
mercaptopurinosuccinate
?
show the reaction diagram
-
-
-
-
?
N6-(1,2-dicarboxyethyl)AMP
?
show the reaction diagram
N6-(1,2-dicarboxyethyl)AMP
fumarate + AMP
show the reaction diagram
phosphoribosylsuccinyl-aminoimidazole carboxamide
phosphoribosylaminoimidazole carboxamide + fumarate
show the reaction diagram
-
-
-
-
?
succino-4-aminopyrazolo[3,4-d]pyrimidine ribose 5'-phosphate
4-aminopyrazolo[3,4-d]pyrimidine ribose 5'-phosphate + ?
show the reaction diagram
-
-
-
?
succinyladenosine monophosphate
AMP + fumarate
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
(S)-2-[5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido]succinate
fumarate + 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide
show the reaction diagram
-
-
-
-
?
(S)-2-[5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido]succinate
fumarate + 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamido
show the reaction diagram
1-(5-phosphoribosyl)-4-(N-succinocarboxamide)-5-aminoimidazole
5'-phosphoribosyl-5-amino-4-imidazolecarboxamide + fumarate
show the reaction diagram
-
-
-
-
?
4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide
5-aminoimidazole-4-carboxamide ribonucleotide + fumarate
show the reaction diagram
5-aminoimidazole-(N-succinylocarboxamide) ribotide
5-aminoimidazole-4-carboxamide ribotide + fumarate
show the reaction diagram
5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide
5-aminoimidazole-4-carboxamide ribonucleotide + fumarate
show the reaction diagram
adenylosuccinate
AMP + fumarate
show the reaction diagram
N6-(1,2-dicarboxyethyl)AMP
?
show the reaction diagram
N6-(1,2-dicarboxyethyl)AMP
fumarate + AMP
show the reaction diagram
phosphoribosylsuccinyl-aminoimidazole carboxamide
phosphoribosylaminoimidazole carboxamide + fumarate
show the reaction diagram
-
-
-
-
?
succinyladenosine monophosphate
AMP + fumarate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
-
stimulates up to maximum concentration of 80 mM, inhibition at higher concentration
PO43-
-
activates at low concentration, inhibits at high concentrations above 25 mM
SO42-
-
activates at low concentrations, inhibits at concentrations above 25 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2'-deoxy-AMP
4-aminopyrazolo[3,4-d]pyrimidine ribose 5'-phosphate
-
-
5-aminoimidazole-4-carboxamide ribonucleoside
50% inhibition at 0.167 mM
5-aminoimidazole-4-carboxamide riboside 5'-monophosphate
-
i.e. ZMP or AICAR, product inhibition, accumulation in the brain of Lesch-Nyhan disease patients induces inhibition of mitochondrial oxidative phosphorylation and adenylosuccinate lyase activity, followed by dowregulation of ATP levels and multifocal cell death in the cerebellum
8-Aza-AMP
-
-
adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate
adenyloethylphosphonate
-
-
adenylophosphonobutyrate
-
-
-
adenylosuccinate 2',3'-acyclic (N,N-ethyl) diamine
-
weak
adenylosuccinate 2',3'-acyclic dialcohol
-
-
adenylosuccinate 2',3'-acyclic dialdehyde
-
-
ADP
-
weak, competitive
ammonium salt of N6-malonyl adenosine 5'-phosphate
-
noncompetitive inhibitor, irreversible
arabinosyl-AMP
ATP
-
weak, competitive
CuSO4
-
complete inhibition at 0.05 mM
DL-3-phosphonoalanine
-
-
EDTA
-
above 15 mM
erythro-beta-fluoro-4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide
-
-
erythro-beta-fluoroadenylosuccinate
-
-
erythro-beta-fluoroaspartate
-
-
fumarate
I-
-
100-200 mM
KBr
presence of increasing concentrations of KBr of 0.12.5 M disrupt electrostatic interactions leading to ASL dissociation and loss of catalytic activity
KCl
-
stimulates up to maximum concentration of 80 mM, inhibition at higher concentration
mercaptopurinosuccinate
-
substrate inhibition
Mg2+
-
concentration of Mg2+ approaching that of EDTA
N-(5-amino-1-(beta-D-ribofuranosyl)imidazole-4-carbonyl)-L-threo-beta-methylaspartic acid 5'-phosphate
-
-
-
N6-(2-carboxyethyl)-AMP
-
i.e. adenylopropionate
N6-(D-1,2-dicarboxyethyl)-AMP
-
i.e. D-adenylosuccinate
N6-(D-1,2-dicarboxypropyl)-AMP
-
i.e. adenyloglutarate
-
N6-(DL-1,2-dicarboxy-threo-2-hydroxyethyl)-AMP
-
i.e. adenylomalate
N6-(DL-1-carboxy-2-phosphonoethyl)AMP
-
i.e. adenylophosphonopropionate
N6-(L-1-carboxy-2-cyanoethyl)-AMP
-
i.e. adenylocyanopropionate
N6-(L-1-carboxy-2-methylsulfoxyethyl)-AMP
-
i.e. adenylomethylsulfoxypropionate
N6-(L-1-carboxy-2-nitroethyl)-AMP
-
competitive
N6-(L-1-carboxy-2-sulfinoethyl)-AMP
-
i.e. adenylosulfinopropionate
N6-(L-1-carboxy-2-sulfoethyl)-AMP
-
i.e. adenylosulfopropionate
nitroacrylate
-
0.02 mM, 70% inactivation
NO3-
-
100-200 mM
nonenal
phosphate
-
competitive
phosphopropionoadenosine
-
-
-
PO43-
-
activates at low concentration, inhibits at high concentrations above 25 mM
SO42-
-
activates at low concentrations, inhibits at concentrations above 25 mM
threo-beta-4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide
-
-
-
threo-beta-fluoroadenylosuccinate
-
-
trans-4-hydroxy-2-nonenal
virazole 5'-phosphate
-
-
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0437
2',3'-dideoxyadenylosuccinate
-
-
0.0008 - 0.003
2'-deoxy-succino-AMP
0.001 - 0.0081
4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide
0.0003 - 0.0029
5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide
0.0095
8-aza-succino-AMP
-
-
0.00146 - 0.023
adenylosuccinate
0.0013 - 0.0551
adenylsuccinate
0.004 - 0.048
AMP
0.0027 - 0.0056
arabinosyl-succino-AMP
0.035 - 0.76
fumarate
0.02
mercaptopurinosuccinate
-
-
0.0012 - 0.013
N6-(1,2-dicarboxyethyl)AMP
0.0007 - 0.009
phosphoribosylsuccinyl-aminoimidazole carboxamide
0.011
succino-4-aminopyrazolo[3,4-d]pyrimidine ribose 5'-phosphate
-
-
0.0014 - 0.2042
succinyladenosine monophosphate
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00048 - 1.74
adenylosuccinate
2.9
AMP
0.1 - 7.5
succinyladenosine monophosphate
additional information
additional information
Homo sapiens
-
pH-dependence of turnover-numbers
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3640 - 50900
phosphoribosylsuccinyl-aminoimidazole carboxamide
0.5 - 25200
succinyladenosine monophosphate
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16 - 0.21
adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate
0.093
AMP
pH 7.4, 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.000083
-
mutant R310Q, pH 7.0
0.004
mutant S295A, pH 7.0
0.008
mutant H171N, pH 7.0
0.009
mutant H171A, pH 7.0
0.031
mutant H171A, pH 8.5
0.063
-
mutant I62D/D65R, adenylsuccinate, 50 mM HEPES, pH 7.0, 25C
0.068
-
complementation of inactive mutant ASLs, maximum specific activity, T93A + H141Q, 3.0% wild-type activity
0.069
-
mutant I62D/D65R, succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
0.072
-
mutant I62D/D65R/D69E, adenylsuccinate, 50 mM HEPES, pH 7.0, 25C
0.094
-
mutant I62D/D65R/D69E, succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
0.096
-
complementation of inactive mutant ASLs, maximum specific activity, S306A + H141Q, 4.6% wild-type activity
0.1
-
mutant R310K, pH 7.0
0.18
-
mutant I62D/D65R + I62E/D65R/D69E, adenylsuccinate, 50 mM HEPES, pH 7.0, 25C
0.2
wild-type enzyme at 4 mg/ml and 4C
0.21
-
complementation of inactive mutant ASLs, maximum specific activity, T93A + K268Q, 10.1% wild-type activity
0.32
-
complementation of inactive mutant ASLs, maximum specific activity, S306A + K268Q, 15.4% wild-type activity; complementation of inactive mutant ASLs, maximum specific activity, T140A + K268Q, 15.4% wild-type activity
0.33
-
mutant I62E/D65R/D69E, adenylsuccinate, 50 mM HEPES, pH 7.0, 25C
0.46
-
mutant I62E/D65R/D69E, succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
0.53
-
mutant I62E/D65R/D69E, succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
0.54
-
mutant I62E/D65R, adenylsuccinate, 50 mM HEPES, pH 7.0, 25C
0.97
-
mutant I62E/D65R/D69E, adenylsuccinate + succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
1
-
mutant R396H, pH 7.4, 25C
1.07
-
adenylsuccinate and succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
1.09
-
mutant I62D/D65R, adenylsuccinate + succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
1.1
-
mutant R396H, after treatment with guanidiniumHCl, pH 7.4, 25C
1.29
-
mutant I62D/D65R + I62E/D65R/D69E, adenylsuccinate + succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
1.3
-
mutant I62D/D65R/D69E, adenylsuccinate + succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
1.4
-
mutant I62E/D65R/D69E, adenylsuccinate + succinylaminoimidazole carboxamide, 50 mM HEPES, pH 7.0, 25C
1.58
-
adenylsuccinate, 50 mM HEPES, pH 7.0, 25C
1.6
-
mutant R396C, after treatment with guanidiniumHCl, pH 7.4, 25C
1.75
-
wild-type enzyme, pH 7.0
1.8
-
mutant R396C, pH 7.4, 25C
2.4
-
-
3.6
-
final purified human enzyme; imidazole gradient elution
3.8
-
non tagged enzyme
4.65
wild-type protein, pH 7.0
6.4
-
purified recombinant His-tagged mutant R426H enzyme, substrate phosphoribosylsuccinyl-aminoimidazole carboxamide, pH 7.4, 25C
6.7
-
purified recombinant His-tagged mutant R426H enzyme, substrate succinyladenosine monophosphate, pH 7.4, 25C
7.5
-
His-tagged mutant L311V, after treatment with guanidiniumHCl, pH 7.4, 25C
7.8
-
His-tagged mutant L311V, pH 7.4, 25C
8.3
-
His-tagged wild-type, after treatment with guanidiniumHCl, pH 7.4, 25C
8.4
-
wild-type, after treatment with guanidiniumHCl, pH 7.4, 25C
8.5
-
His-tagged wild-type, pH 7.4, 25C
8.6
-
wild-type, pH 7.4, 25C
8.7
-
His-tagged mutant R194C, pH 7.4, 25C
8.9
-
His-tagged mutant R194C, after treatment with guanidiniumHCl, pH 7.4, 25C
13.9
-
purified recombinant His-tagged wild-type enzyme, substrate succinyladenosine monophosphate, pH 7.4, 25C
16.47
wild-type protein, pH 8.5
20
-
crude cell extract, presence of 1 M KCl, pH 7.5, temperature not specified in the publication
23.3
-
purified recombinant His-tagged wild-type enzyme, substrate phosphoribosylsuccinyl-aminoimidazole carboxamide, pH 7.4, 25C
40
-
crude cell extract, presence of 1 M KCl, pH 7.5, temperature not specified in the publication
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.9
-
synthesis of N6-(1,2-dicarboxyethyl)AMP
7.7 - 8.7
-
Tris buffer
7.8
-
all cell lines examined
8 - 8.5
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9.5
-
inactive below pH 6 and above 9.5
7.2 - 8.4
-
-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus subtilis (strain 168)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Francisella tularensis subsp. tularensis (strain SCHU S4 / Schu 4)
Legionella pneumophila subsp. pneumophila (strain Philadelphia 1 / ATCC 33152 / DSM 7513)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
Plasmodium vivax (strain Salvador I)
Plasmodium vivax (strain Salvador I)
Pyrobaculum aerophilum (strain ATCC 51768 / IM2 / DSM 7523 / JCM 9630 / NBRC 100827)
Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720)
Staphylococcus aureus (strain MW2)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Thermotoga maritima (strain ATCC 43589 / MSB8 / DSM 3109 / JCM 10099)
Trypanosoma brucei brucei (strain 927/4 GUTat10.1)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
57000
-
histidine-tagged enzyme, determined by SDS-PAGE
147000
-
determined by native polyacrylamide gel electrophoresis, wild-type enzyme
148000
-
determined by native polyacrylamide gel electrophoresis, mutant R310K
152000
-
mutant N270L, light scattering measurements, 0.2 mg/ml enzyme in a filtered 20 mM potassium phosphate buffer at pH 7.0, containing 20 mM potassium chloride
153000
-
wild type, light scattering measurements, 0.2 mg/ml enzyme in a filtered 20 mM potassium phosphate buffer at pH 7.0, containing 20 mM potassium chloride
155000
-
mutant D69E, light scattering method, 0.25 mg/ml
159000
166000
-
mutant N270D, light scattering measurements, 0.2 mg/ml enzyme in a filtered 20 mM potassium phosphate buffer at pH 7.0, containing 20 mM potassium chloride
173000
174000
-
mutant Q212M, light scattering measurements, 0.2 mg/ml enzyme in a filtered 20 mM potassium phosphate buffer at pH 7.0, containing 20 mM potassium chloride
176000
181000
-
light scattering method, 0.25 mg/ml
182000
-
determined by light scattering, mutant R310K
191000
192000
-
mutant S263H, light scattering
193000
-
determined by native polyacrylamide gel electrophoresis, mutant S93A
194000
194800
-
determined by analytical ultracentrifugation, mutant T140A
196000
197000
-
mutant S263A, light scattering
199000
-
mutant S262H, light scattering
199700
-
determined by analytical ultracentrifugation, wild-type enzyme
200000
201000
-
mutant enzyme S413P, gel filtration
209000
-
mutant I62E/D65R, native PAGE
210000
-
native PAGE
214500
-
recombinant His-tagged R303C mutant enzyme
216400
-
determined by analytical ultracentrifugation, mutant S306A
217000
-
mutant I62D/D65R, native PAGE
219000
-
mutant R301Q, light scattering measurements, 0.2 mg/ml enzyme in a filtered 20 mM potassium phosphate buffer at pH 7.0, containing 20 mM potassium chloride
221000
-
mutant S290H, light scattering
222000
-
analytical ultracentrifugation
223000
-
gel filtration, at 22C
225000
-
recombinant His-tagged wild-type enzyme
227000
228000
-
gel filtration, His-tagged protein
230000
-
analytical ultracentrifugation, at 25C
234000
237000
-
mutant S289A, light scattering
300000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
-
2 * 50000, SDS-PAGE
homotetramer
tetramer
additional information
-
enzyme is composed of 6-8 identical subunits
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 1.4 M sodium/potassium phosphate pH 7.5 and 4% (v/v) glycerol
the crystal structures of wild-type ADL, and mutant-substrate, H171A-ADS, and -product, H171N-AMP-FUM, complexes are determined to 2.0, 1.85, and 2.0 A resolution, respectively
purified wild-type apo-enzyme and enzyme mutant R303C, high throughput screening by sitting drop method, method optimization, mixing of 0.004 ml of protein solution with 0.004 ml of reservoir solution and equilibration against 0.5 ml of reservoir solution, with a precipitant gradient for the native enzyme of 18-28% w/v PEG 6000, 0.1 M Tris, pH 8.0, and 12.5 mM MgCl2 hexahydrate, and for enzyme mutant R303C, a precipitant gradient of 18-28% w/v PEG 8000, 0.1 M Tris, pH 8.5, and 12.5 mM spermine tetrahydrochloride, X-ray diffraction structure determination and analysis at 2.7 A and 2.6 A resolution, respectively
-
purified enzyme, X-ray diffraction structure determination and analysis at 2.16 A resolution
purified enzyme, X-ray diffraction structure determination is not possible
-
at a resolution of 2.1 A
-
crystal structure is determined to 2.1 resolution. Hanging drop vapor diffusion method at 18C
in complex with AMP, at 2.5 A resolution, and modeling of oxalate in the active site. Comparisons with the enzyme from Escherichia coli and with human PurB show close similarity of the active sites, but differences in the way that the subunits are assembled into dimers
at a resolution of 1.8 A
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
enzyme is stable for at least 5 days at 25C, and upon rapid freezing and thawing
37
-
t1/2 for enzyme from mutant cell line 1751: 0.23 min, t1/2 for enzyme from mutant cell line 1753: 0.2 min, t1/2 for enzyme from the normal cell lines: 9.65 h
50
-
at 50C the wild type enzyme shows 79-84% of the activity at 25C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
dialysis for 24 h at 5C, activity in mutant lysates is decreased by 95% while 50-60% of the initial activity remains in the normal cell lysates
-
freezing at -80C, followed by thawing, 20-40% loss of enzyme activity in extract
-
stable to thawing and refreezing at a protein concentration of above 2 mg/ml
-
the structure of adenylosuccinate lyase reveals that this protein contains three disulfide bonds that contribute significantly to its stability against thermal and chemical denaturation
three cycles of freezing and thawing remove nearly all enzyme activity
-
unstable to quick freezing in dry ice/acetone
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, 10% glycerol, 1 mM dithiothreitol, 0.01 M Tris, pH 7.4, activity in lysates from all cell lines is stable for more than 6 weeks
-
-70C, unstable to storage
-
-80C or -20C, recombinant enzyme is very unstable regardless of salt or glycerol concentration
-
-80C, potassium phosphate buffer, KCl, DTT, EDTA, glycerol
-
2C or -20C, stable for two months
-
4C, stable for several weeks
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-affinity chromatography
-
Ni-NTA agarose column chromatography
-
normal and mutant enzyme S413P
-
Q-Sepharose column chromatography and gel filtration
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta 2(DE3)pLysS by nickel affinity chromatography
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta 2(DE3)pLysS by nickel affinity chromatography, dialysis, cleavage of the His-tag through thrombin, and gel filtration, to homogeneity
-
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain Bl21 by nickel affinity chromatography
using a Ni-NTA column
using a Ni-NTA column, the 6-histidine tag is removed by cleavage with thrombin, separation of thrombin and histidine-tag from the enzyme is achieved by using a Sephadex G-100 column
-
using nickel affinity chromatography
-
using nickel nitrilotriacetic acid-agarose
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ADSL gene, genotyping in Polish patients with adenylosuccinate lyase deficiency compared to the wild-type, overview
-
expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli DH5alphaF'IQ cells as recombinant maltose binding protein-ADSL fusion protein
-
expressed in Escherichia coli Rosetta 2 (DE3) cells
-
expression in Escherichia coli
expression in Escherichia coli as His tag enzymes
-
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta 2(DE3)pLysS
-
expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21
expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta 2(DE3)pLysS
-
expression of wild-type and mutant enzymes in Escherichia coli strain DH5alpha
for expression in Escherichia coli BL21DE3 cells
-
for expression in Escherichia coli strain BL21DE3
-
into the pET-14b vector for expression in Escherichia coli Rosetta 2DE3pLysS cells
-
normal and mutant enzyme S413P, expression in Escherichia coli
-
phylogenetic analysis
subcloned from the pUC118 into the pET28a vector for expression in Escherichia coli B834DE3 cells
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D65R
-
mutant with differences in thermal stability, catalytic activity and substrate binding
D65R/D69E
-
mutant with differences in thermal stability, catalytic activity and substrate binding
E239M
site-directed mutagenesis, the mutant shows reduced Vmax, increased Km, and lower molecular weight compared to the wild-type enzyme, subunit structure analysis
E239Q
site-directed mutagenesis, the mutant shows reduced Vmax, increased Km, and lower molecular weight compared to the wild-type enzyme, subunit structure analysis
E239R
site-directed mutagenesis, the mutant shows reduced Vmax, increased Km, and lower molecular weight compared to the wild-type enzyme, subunit structure analysis
H141Q
-
site-directed mutagenesis, the mutant shows altered binding of inhibitory substrate analogue adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate compared to the wild-type enzyme
H299K
site-directed mutagenesis, the mutant shows reduced Vmax, increased Km, and lower molecular weight compared to the wild-type enzyme, subunit structure analysis
H299R
site-directed mutagenesis, the mutant shows reduced Vmax, increased Km, and lower molecular weight compared to the wild-type enzyme, subunit structure analysis
H68A
-
site-directed mutagenesis, the mutant shows altered binding of inhibitory substrate analogue adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate compared to the wild-type enzyme
H89Q
-
site-directed mutagenesis, the mutant shows altered binding of inhibitory substrate analogue adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate compared to the wild-type enzyme
H89R
-
site-directed mutagenesis, the mutant shows altered binding of inhibitory substrate analogue adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate compared to the wild-type enzyme
I62D
-
mutant with differences in thermal stability, catalytic activity and substrate binding
I62D/D65R
-
mutant with differences in thermal stability, catalytic activity and substrate binding
I62D/D65R/D69E
-
mutant with differences in thermal stability, catalytic activity and substrate binding
I62D/D69E
-
mutant with differences in thermal stability, catalytic activity and substrate binding
I62E
-
mutant with differences in thermal stability, catalytic activity and substrate binding
I62E/D65R
-
'humanized' normal enzyme
I62E/D65R/D69E
-
mutant with differences in thermal stability, catalytic activity and substrate binding
I62E/D69E
-
mutant with differences in thermal stability, catalytic activity and substrate binding
N270D
-
mutant with impaired activity
N270L
-
mutant with impaired activity
Q212E
-
mutant with impaired activity
Q212M
-
mutant with impaired activity
R167E
site-directed mutagenesis, the mutant shows reduced Vmax, increased Km, and lower molecular weight compared to the wild-type enzyme, subunit structure analysis
R167Q
site-directed mutagenesis, the mutant shows reduced Vmax, increased Km, and lower molecular weight compared to the wild-type enzyme, subunit structure analysis
R301K
-
mutant with impaired activity
R301Q
-
mutant with impaired activity
R310K
-
mutant shows little change in pK2
R310Q
-
mutant exibits a decrease in its pK2
S262A
-
site-directed mutagenesis, the mutant shows no activity and altered molecular weight compared to the wild-type enzyme
S262H
-
site-directed mutagenesis, the mutant shows no activity and altered molecular weight compared to the wild-type enzyme
S263A
-
site-directed mutagenesis, the mutant shows no activity and altered molecular weight compared to the wild-type enzyme
S263H
-
site-directed mutagenesis, the mutant shows no activity and altered molecular weight compared to the wild-type enzyme
S306A
-
conserved amino acid in all adenylosuccinate lyases, mutant for testing the involvement of the residue in the enzyme function
S94A
-
conserved amino acid in all adenylosuccinate lyases, mutant for testing the involvement of the residue in the enzyme function
T140A
-
conserved amino acid in all adenylosuccinate lyases, mutant for testing the involvement of the residue in the enzyme function
T93A
-
conserved amino acid in all adenylosuccinate lyases, mutant for testing the involvement of the residue in the enzyme function
H171A
mutant to map out the residues involved in adenylosuccinate binding, and to identify the putative catalytic groups
H171N
mutant to map out the residues involved in adenylosuccinate binding, and to identify the putative catalytic groups
S295A
mutant to probe the role of S295 in the catalytic mechanism
A219V
-
site-directed mutagenesis, inactive mutant
A260GfsX24
-
the mutation is associated with ADSL deficiency
D422Y
-
mutant without enzyme activity
D87E
-
the mutation is associated with ADSL deficiency and shows 91% activity using 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide as substrate at 25C compared to the wild type enzyme
E376D
-
the mutation is associated with ADSL deficiency and shows 49% activity using 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide as substrate at 25C compared to the wild type enzyme
E80D
-
the mutation is associated with ADSL deficiency and shows 102% activity using 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide as substrate at 25C compared to the wild type enzyme
E80D/D87E
-
australian patient with autism
K246E
-
the mutation is associated with adenylosuccinate lyase deficiency, the Vmax for the K246E mutant enzyme is about 2% that of the wild type enzyme, K246E exists mainly as dimer or monomer
L423V
-
mutant without enzyme activity
P100A/D422Y
-
the mutation leads to a defective enzyme which is inhibited by micromolar concentrations of trans-4-hydroxy-2-nonenal
P110A/D422Y
-
heat sensitive mutant
R141W
-
thermostable mutant with decreased activity
R149G
-
the mutation is associated with ADSL deficiency
R190Q
-
the mutation is associated with ADSL deficiency and shows 119% activity using 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide as substrate at 25C compared to the wild type enzyme
S289A
-
site-directed mutagenesis, the mutant shows no activity, and altered molecular weight and binding of inhibitory substrate analogue adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate compared to the wild-type enzyme
S289H
-
site-directed mutagenesis, the mutant shows no activity, and altered molecular weight and binding of inhibitory substrate analogue adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate compared to the wild-type enzyme
S290A
-
site-directed mutagenesis, the mutant shows altered molecular weight and binding of inhibitory substrate analogue adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate compared to the wild-type enzyme
S290H
-
site-directed mutagenesis, the mutant shows no activity, and altered molecular weight and binding of inhibitory substrate analogue adenosine phosphonobutyric acid, 2'(3'),5'-diphosphate compared to the wild-type enzyme
S395R
-
thermostable mutant with decreased activity
S413P
-
the mutation leads to structural instability of the mutant enzyme, this instability lowers the enzyme level in lymphocytes. The mutation segregates with mental retardation in the affected family
S438P
-
heat sensitive mutant without enzyme activity
T450S
-
the mutation is associated with ADSL deficiency and shows 68% activity using 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide as substrate at 25C compared to the wild type enzyme
S298A
site-directed mutagenesis, the mutation does not lead to gross changes in the structural properties of ASL, but the specific activity of S298A mutant is 1000fold lower than that of the wild enzyme, the specific activity remains unchanged upon variation of pH
S298C
site-directed mutagenesis, the mutation does not lead to gross changes in the structural properties of ASL, but the activity of the S298C mutant is completely lost
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
additional information
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