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Information on EC 2.3.1.37 - 5-aminolevulinate synthase and Organism(s) Mus musculus and UniProt Accession P08680
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2.3.1.37 5-aminolevulinate synthaseIUBMB Comments
A pyridoxal-phosphate protein. The enzyme in erythrocytes is genetically distinct from that in other tissues.
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Word Map
- 2.3.1.37
- heme
- porphyria
-
sideroblastic
- oxygenase
- anemia
-
x-linked
-
ferrochelatase
- hemoglobin
- marrow
- porphobilinogen
- dehydratase
- pyridoxal
- uroporphyrinogen
- protoporphyria
-
iron-responsive
- hemoprotein
- erythroblast
- phenobarbital
-
erythropoietic
- tetrapyrrole
- rhodopseudomonas
- rhodobacter
-
microcytic
-
porphyrinogenic
- erythroleukemia
-
delta-aminolaevulinate
-
allylisopropylacetamide
-
uroporphyrin
- medicine
- synthesis
-
hypochromic
- pharmacology
- gata1
- aip
- hexachlorobenzene
-
plp-dependent
-
cutanea
-
5'-phosphate-dependent
-
phenobarbital-induced
- biotechnology
-
neurovisceral
- nutrition
-
rate-controlling
-
pyrrolase
- protoporphyrinogen
-
heme-mediated
- tarda
-
5-aminolaevulinate
-
succinyl-coenzyme
- bacteriochlorophyll
- coproporphyrinogen
- agriculture
-
harderian
- succinylacetone
The taxonomic range for the selected organisms is: Mus musculus
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
alas2, ala synthase, 5-aminolevulinate synthase, ala-s, delta-aminolevulinate synthase, delta-aminolevulinic acid synthase, delta-aminolevulinic acid synthetase, 5-aminolevulinic acid synthase, aminolevulinate synthase, delta-aminolevulinate synthetase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
ALAS2
5-aminolevulinate synthetase
-
-
-
-
5-aminolevulinic acid synthetase
-
-
-
-
ALA synthase
-
-
-
-
ALA synthetase
-
-
-
-
ALAS
alpha-aminolevulinic acid synthase
-
-
-
-
aminolevulinate synthase
-
-
-
-
aminolevulinate synthetase
-
-
-
-
aminolevulinic acid synthase
-
-
-
-
aminolevulinic acid synthetase
-
-
-
-
aminolevulinic synthetase
-
-
-
-
delta-aminolevulinate synthase
-
-
-
-
delta-aminolevulinate synthetase
-
-
-
-
delta-aminolevulinic acid synthase
-
-
-
-
delta-aminolevulinic acid synthetase
-
-
-
-
delta-aminolevulinic synthetase
-
-
-
-
mALAS-2
-
mature form of the murine erythroid specific isoform of aminolevulinate synthase
synthase, aminolevulinate
-
-
-
-
synthetase, aminolevulinate
-
-
-
-
ALAS
-
-
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
proposed chemical mechanism of enzyme ALAS2 via (I) internal aldimine complex, (II) glycine-external aldimine, (III) quinonoid intermediate I, (IV) glycine-succinyl-CoA condensation intermediate, (V) 2-amino-3-ketoadipate intermediate, (VI) enol intermediate, (VII) quinonoid intermediate II, and (VIII) 5-aminolevulinate-external aldimine
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
proposed chemical mechanism of enzyme ALAS2. via (I) internal aldimine complex, (II) glycine-external aldimine, (III) quinonoid intermediate I, (IV) glycinesuccinyl-CoA condensation intermediate, (V) 2-amino-3-ketoadipate intermediate, (VI) enol intermediate, (VII) quinonoid intermediate II, and (VIII) 5-aminolevulinate-external aldimine
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
the rate of 5-aminolevulinate release is controlled by a hysteretic kinetic mechanism initiated by conformational changes of the enzyme. The active site residue Thr148 modulates the enzyme's strict amino acid substrate specificity. Catalytic mechanism, overview
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
covalent binding of pyridoxal 5'-phosphate and glycine to active site Lys131 is required for optimal activity
-
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
Tyr121, Asp279, Arg439, and Lys313 are involved in substrate and cofactor binding, mechanism, subunit localisation
-
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
cysteine in heme-regulatory motif
-
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
Lys313 acts as a general base during formation of the quinonoid reaction intermediates
-
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
the active site is located at the subunit interface and contains catalytically essential residues from the two subunits
-
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
three-step kinetic process, ordered kinetic mechanism, reaction mechanism
-
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
pyridoxal 5'-phosphate binding site, sequence and function of glycine-rich motif
-
succinyl-CoA + glycine = 5-aminolevulinate + CoA + CO2
5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathans hypothesis
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Acyl group transfer
-
-
-
-
PATHWAY SOURCE
PATHWAYS
BRENDA
KEGG
-
-, -
SYSTEMATIC NAME
IUBMB Comments
succinyl-CoA:glycine C-succinyltransferase (decarboxylating)
A pyridoxal-phosphate protein. The enzyme in erythrocytes is genetically distinct from that in other tissues.
CAS REGISTRY NUMBER
COMMENTARY
9037-14-3
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
succinyl-CoA + L-serine
?
the reaction with L-serine follows a biphasic kinetic process
-
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
it is the CoA, rather than the succinyl moiety, that facilitates binding of succinyl-CoA to wild-type ALAS
-
-
r
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
-
-
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
key enzyme in tetrapyrrole biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
cellular iron status plays a regulatory role
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
regulatory mechanisms in hepatic and erythroid cells
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
first step in heme biosynthesis pathway
-
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
first step in heme biosynthesis pathway
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
first step in heme biosynthesis pathway
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
5-aminolevulinate synthase operates under the stereoelectronic control predicted by Dunathans hypothesis
-
-
?
additional information
?
-
analysis of unstable enzyme-catalyzed reaction intermediates and conformational changes using physiological and non-physiological substrates and promiscuous T148A enzyme variant, overview. Formation of the quinonoid intermediate upon reacting glycine with the enzyme. Significantly, in the absence of the succinyl-CoA substrate, the external aldimine predominates over the glycine quinonoid intermediate. When instead of glycine, L-serine is reacted with the enzyme, a lag phase is observed in the progress curve for the L-serine external aldimine formation, indicating a hysteretic behavior in enzyme ALAS. Hysteresis is not detected in the T148A-catalyzed L-serine external aldimine formation. The rate of 5-aminolevulinate release is also controlled by a hysteretic kinetic mechanism. The active site residue Thr148 modulates the enzyme's strict amino acid substrate specificity, positioning of the glycine external aldimine in the active site, overview
-
-
?
additional information
?
-
-
analysis of unstable enzyme-catalyzed reaction intermediates and conformational changes using physiological and non-physiological substrates and promiscuous T148A enzyme variant, overview. Formation of the quinonoid intermediate upon reacting glycine with the enzyme. Significantly, in the absence of the succinyl-CoA substrate, the external aldimine predominates over the glycine quinonoid intermediate. When instead of glycine, L-serine is reacted with the enzyme, a lag phase is observed in the progress curve for the L-serine external aldimine formation, indicating a hysteretic behavior in enzyme ALAS. Hysteresis is not detected in the T148A-catalyzed L-serine external aldimine formation. The rate of 5-aminolevulinate release is also controlled by a hysteretic kinetic mechanism. The active site residue Thr148 modulates the enzyme's strict amino acid substrate specificity, positioning of the glycine external aldimine in the active site, overview
-
-
?
additional information
?
-
the wild-type enzyme catalyzes the conversion of 5-aminolevulinate into the quinonoid intermediate at a rate 6.3fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA. The mutant N150F enzyme catalyzes the forward reaction at a 1.2fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7fold faster rate for the reverse reaction than that for the forward reaction. Steric constraints imposed by the active site of wild-type enzyme ALAS contribute toward the amino acid substrate specificity
-
-
?
additional information
?
-
-
the wild-type enzyme catalyzes the conversion of 5-aminolevulinate into the quinonoid intermediate at a rate 6.3fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA. The mutant N150F enzyme catalyzes the forward reaction at a 1.2fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7fold faster rate for the reverse reaction than that for the forward reaction. Steric constraints imposed by the active site of wild-type enzyme ALAS contribute toward the amino acid substrate specificity
-
-
?
additional information
?
-
no activity with L-alanine, L-serine or L-threonine
-
-
-
additional information
?
-
-
no activity with L-alanine, L-serine or L-threonine
-
-
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
key enzyme in tetrapyrrole biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
cellular iron status plays a regulatory role
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
regulatory mechanisms in hepatic and erythroid cells
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
first step in heme biosynthesis pathway
-
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
first step in heme biosynthesis pathway
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
first step in heme biosynthesis pathway
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
succinyl-CoA + glycine
5-aminolevulinate + CoA + CO2
-
rate-limiting enzyme of heme biosynthesis
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
pyridoxal 5'-phosphate
-
required
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Iron
-
regulation of erythroid-specific isoform ALAS-2 via 5'-iron responsive element, i.e. IRE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
hexamethylene diacetamide
-
induction of enzyme expression, erythroleukemia cells
O2
-
1%, 3fold increase in expression level, promotor activation in transiently transfected HeLa cells, hypoxia-inducible erythroid-isoform
additional information
-
iron depletion does not increase isoform II promotor activity
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.0922
glycine
-
mutant cosubstrate 2-hydroxybutanoyl-CoA, R85L/T430V, pH 7.5, 30°C
additional information
additional information
Michaelis-Menten steady-state kinetics, overview
-
additional information
additional information
kinetics analysis of wild-type and mutant enzymes, single and multiple turnover and stopped flow measurements, substrate protection study, overview
-
additional information
additional information
-
kinetics analysis of wild-type and mutant enzymes, single and multiple turnover and stopped flow measurements, substrate protection study, overview
-
additional information
additional information
pre-steady state and steady state kinetics, overview
-
additional information
additional information
-
pre-steady state and steady state kinetics, overview
-
additional information
additional information
pre-steady-state and steady-state kinetics of wild-type and mutant enzymes, stopped-flow measurements, single and mutiple turnover rates, equilibrium dissociation constants, binding isotherms, detailed overview
-
additional information
additional information
-
pre-steady-state and steady-state kinetics of wild-type and mutant enzymes, stopped-flow measurements, single and mutiple turnover rates, equilibrium dissociation constants, binding isotherms, detailed overview
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.5
7.5
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
ALA-S activity is induced by acute administration of anaesthetics (89%), veronal (240%), and ethanol (80). ALA-S mRNA expression augmented by chronic administration of eflurane, allylisopropylacetamide and veronal
-
including submandibular and parotid glands, high expression of ALAS1
additional information
additional information
-
enzyme is synthezised in the cytosol as the precursor protein and then imported into the mitochondria matrix and cleaved to the mature enzyme, the targeting information is encoded in nonoverlapping regions of the presequence
additional information
-
enzyme is synthezised in the cytosol as the precursor protein and then imported into the mitochondria matrix and cleaved to the mature enzyme, the targeting information is encoded in nonoverlapping regions of the presequence
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
metabolism
physiological function
-
construction of a green fluorescent protein knock-in mouse line in which the Alas1 gene encoding ALAS-N is replaced with a green fluorescent protein gene. Mice bearing a homozygous knock-in allele Alas1GFP /GFP are lethal by embryonic day 8.5. The Alas1 expression level differs substantially in tissues
additional information
malfunction
mutations of the murine ALAS2 active site loop result in increased production of protoporphyrin IX, the precursor for heme. Generation of protoporphyrin IX is a crucial component in the widely used photodynamic therapies of cancer and other dysplasias. ALAS2 variants that cause high levels of protoporphyrin IX accumulation provide a means of targeted, and potentially enhanced, photosensitization. ALAS2-induced protoporphyrin IX accumulation followed by light exposure combined with paclitaxel treatment causes cell death
malfunction
genetic mutations in the erythroid-specific isoform ALAS2 are associated with two inherited blood disorders, X-linked sideroblastic anemia (XLSA) and X-linked protoporphyria (XLPP). XLSA is caused by diminished ALAS2 activity leading to decreased ALA and heme syntheses and ultimately ineffective erythropoiesis, whereas XLPP results from gain-of-function ALAS2 mutations and consequent overproduction of protoporphyrin IX and increase in Zn2+-protoporphyrin levels
metabolism
5-aminolevulinate synthase catalyzes the first committed step of heme biosynthesis in animals
metabolism
5-aminolevulinate synthase catalyzes the initial step of mammalian heme biosynthesis
metabolism
5-aminolevulinate synthase catalyzs the initial step of mammalian heme biosynthesis
metabolism
-
5-aminolevulinate synthase catalyzs the initial step of mammalian heme biosynthesis
additional information
residue Asn150 is essential for establishing a catalytic balance between the forward and reverse reactions
additional information
-
residue Asn150 is essential for establishing a catalytic balance between the forward and reverse reactions
additional information
the adenosyl-binding site Lys221 contributes to binding and orientation of succinyl-CoA for effective catalysis
additional information
-
the adenosyl-binding site Lys221 contributes to binding and orientation of succinyl-CoA for effective catalysis
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). HEM0_MOUSE
587
0
64753
Swiss-Prot
Mitochondrion (Reliability: 5)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
56000
additional information
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
dimer
additional information
-
the active site is located at the subunit interface and contains catalytically essential residues from the two subunits
homodimer
the individual active sites are located at the dimeric interface and are composed of amino acids from each subunit
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
K221V
N150A
site-directed mutagenesis, the mutation significantly reduces the rate of quinonoid intermediate formation in the forward direction
N150F
site-directed mutagenesis, the mutation significantly reduces the rate of quinonoid intermediate formation in the forward direction, while increasing the reverse reaction rate
N150G
site-directed mutagenesis, the mutation significantly reduces the rate of quinonoid intermediate formation in the forward direction
N150H
site-directed mutagenesis, the mutation significantly reduces the rate of quinonoid intermediate formation in the forward direction, while increasing the reverse reaction rate
N150W
site-directed mutagenesis, the mutation significantly reduces the rate of quinonoid intermediate formation in the forward direction
R433K
site-directed mutagenesis, mALAS2 variant with a mutated presequence, the mutation results in an increase in activity to twice that of the wild-type enzyme, i.e. a 2fold increase in the kcat value and a 1.65 to 1.85fold enhancement in the specificity constants for glycine and succinyl-CoA over those of wild-type, mature mALAS2, 2.5fold increase in protoporphyrin IX accumulation in HeLa cells expressing the R433K precursor with a mutated presequence
T148A
site-directed mutagenesis, the active site Thr148 mutation modulates the enzyme's strict amino acid substrate specificity
D279A
-
exchange mutant of potential cofactor binding residue Asp279, no activity, dissociation constant for pyridoxal 5'-phosphate is 19fold increased, different mode of cofactor binding, no formation of quinonoid reaction intermediate, which can be restored by addition of analogue N-methyl-pyridoxal 5'-phosphate
D279E
-
exchange mutant of potential cofactor binding residue Asp279, 30fold reduced catalytic efficiency for succinyl-CoA compared to the wild-type
G142C
-
glycine-rich motif mutant, 15fold increased dissociation constant value for binding of cofactor pyridoxal 5'-phosphate, 6% turnover compared to the wild-type, 4fold increase of Km-value for glycine
G144A
-
glycine-rich motif mutant, 8.5fold increased dissociation constant value for binding of cofactor pyridoxal 5'-phosphate, 43% turnover compared to the wild-type, unaltered Km-values for the substrates
G144S
-
glycine-rich motif mutant, 8fold increased dissociation constant value for binding of cofactor pyridoxal 5'-phosphate, 39% turnover compared to the wild-type, unaltered Km-values for the substrates
G144T
-
glycine-rich motif mutant, 24.5fold increased dissociation constant value for binding of cofactor pyridoxal 5'-phosphate, 21% turnover compared to the wild-type, unaltered Km-values for the substrates
K313A
K313A/R149A
-
each mutation site located on 1 subunit, 2 plasmids, coexpression of the dimer in Escherichia coli hemA-, functional complementation, 26% activity compared to wild-type
K313G
-
site-directed mutagenesis, mutants of erythroid-specific isoform, exchange of active site lysine residue 313, binding of pyridoxal 5'-phosphate and glycine noncovalently, reduced activity, because covalent binding is required
K313H
R149A
-
mutation site located at the active site of 1 subunit, functional complementation of Escherichia coli mutant strain hemA-, no activity
R439K
-
active site mutant, 77% activity compared to the wild-type, 9-13fold increased Km for both substrates, 5fold increased dissociation constant for glycine
R439L
-
active site mutant, no activity, 30fold increased dissociation constant for glycine
S254A
-
increase in Km value for succinyl-Coa and kcat value. Removal of the side chain hydroxyl group alters the microenvironment of the PLP cofactor and hinders succinyl-CoA binding
Y121F
-
exchange mutant of potential cofactor binding residue Tyr121, 5% activity compared to the wild-type, Km for glycine is 5fold increased, lower affinity for pyridoxal 5'phosphate
Y121H
-
exchange mutant of potential cofactor binding residue Tyr121, 36% activity compared to the wild-type, Km for glycine is 34fold increased, lower affinity for pyridoxal 5'phosphate
additional information
K221V
random mutagenesis, library screening, the mutation produces a 23fold increased Km for succinyl-CoA and a 97% decrease in kcat/Km for succinyl-CoA. This reduction in the specificity constant does not stem from lower affinity toward succinyl-CoA, since the Kd for succinyl-CoA of K221V is lower than that of the wild-type enzyme. Mutant K221V has a stronger binding affinity for succinyl-CoA compared to the wild-type enzyme. The mutation reduces the rates of quinonoid intermediate II formation and decay
K221V
the mutant shows severely reduced catalytic efficiency towards succinyl-CoA compared to the wild type enzyme
K313A
-
mutation site located at the active site of 1 subunit, functional complementation of Escherichia coli mutant strain hemA-, no activity
K313A
-
site-directed mutagenesis, mutants of erythroid-specific isoform, exchange of active site lysine residue 313, binding of pyridoxal 5'-phosphate and glycine noncovalently, reduced activity, because covalent binding is required
K313H
-
site-directed mutagenesis, mutants of erythroid-specific isoform, exchange of active site lysine residue 313, binding of pyridoxal 5'-phosphate and glycine noncovalently, reduced activity, because covalent binding is required
additional information
generation of enzyme mutant with mutated mitochondrial presequences, at residues C11 C38, and C70, and a mutation in the active site loop Expression of the mutants in human cells causes significant cellular accumulation of protoporphyrin IX, particularly in the membrane. ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid treatment producing a similar amount of protoporphyrin IX. Supplementation of cell culture medium with glycine leads to increased protoporphyrin IX accumulation in Malas2-expressing HeLa cells
additional information
-
generation of enzyme mutant with mutated mitochondrial presequences, at residues C11 C38, and C70, and a mutation in the active site loop Expression of the mutants in human cells causes significant cellular accumulation of protoporphyrin IX, particularly in the membrane. ALAS2 expression results in an increase in cell death in comparison to aminolevulinic acid treatment producing a similar amount of protoporphyrin IX. Supplementation of cell culture medium with glycine leads to increased protoporphyrin IX accumulation in Malas2-expressing HeLa cells
additional information
selection of functional mALAS2 variants from the Thr148/Asn150 library of constructs is accomplished by reversing the 5-aminolevulinate auxotrophic phenotype of Escherichia coli hemA (HU227) cells
additional information
-
selection of functional mALAS2 variants from the Thr148/Asn150 library of constructs is accomplished by reversing the 5-aminolevulinate auxotrophic phenotype of Escherichia coli hemA (HU227) cells
additional information
the truncation of the N-terminal region in mutant DELTAmALAS2 does not affect the Km values for either substrate while the kcat of the mutant enzyme, relative to that of the wild type enzyme, is increased by 22%
additional information
-
the truncation of the N-terminal region in mutant DELTAmALAS2 does not affect the Km values for either substrate while the kcat of the mutant enzyme, relative to that of the wild type enzyme, is increased by 22%
additional information
-
site-directed mutagenesis of hypoxia-inducible factor-1, i.e. HIF-1, binding site in promotor sequence -328/-318, reveales HIF-1 like activation of enzyme expression during hypoxia
additional information
-
stable MEL mutant, no induction of enzyme expression by dimethylsulfoxide, hexamethylene diacetamide and butyric acid, decline of enzyme activity after DMSO application
additional information
-
expression of erythroid-specific isoform in MEL mutant under control of metallothionin promotor results in induction of enzyme activity by addition of Zn2+ and Cd2+ in absence of DMSO
additional information
-
circularly permuted enzyme variants with N-terminal amino acids corrosponding to L25, Q69, N404, N408 and a monomeric protein consisting of two wild-type enzyme subunits covalently linked through the N-terminus of one subunit to the C-terminuns of the other. Analysis of guanidine hydrochloride-induced unfolding, conformational stability, and structure
additional information
-
linkage of two subunits into a single polypeptide chain dimer 2XALAS, results in enzyme with about 7fold greater turnover number than wild-type and with greater A410/A330 ratio
additional information
-
a single chain dimeric ALAS variant is created, in which one of the two active sites harbors a K313A mutation eliminating measurable enzyme activity in order to investigate the unusual enhanced enzymatic activity resulting from linking ALAS dimmers. The two active sites in ALAS/ALAS differentially contribute to the enhanced activity of the enzyme, even though the amount of ALA produced during the first turnover is identical in both active sites. The kcat values of the K313A variants differ significantly depending on which of the two active sites harbors the mutation
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
the secondary structure of the enzyme is mostly resilient to pH changes. Partial unfolding is observed at pH 2.0, but further decreasing pH results in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity is lost under acidic and specific alkaline conditions, pH 10.5 and pH 9.5 at 37°C, where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified. Under acidic conditions, the pyridoxal 5-phosphate cofactor dissociates from the enzyme. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, far-UV and near-UV circular dichroism study, detailed overview. The alkaline molten globule state of ALAS is catalytically active
735711
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
the secondary structure of the enzyme is mostly resilient to temperature changes, overview
additional information
-
thermal stability of glycine-rich motif mutants is reduced compared to the wild-type
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
in the presence of denaturing guanidine hydrochloride concentrations at pH 10.5, the rate of enzyme denaturation is 3times faster than at pH 7.5. Both guanidine hydrochloride- and temperature-induced denaturation reduce the enzyme stability at pH 10.5
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant wild-type and mutant enzymes
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene ALAS, recombinant expression in HeLa cells, stable expression of enzyme variants in K-562 cells
recombinant expression of wild-type and mutant enzymes
expression of erythroid-specific isoform ALAS-E and mutants in Escherichia coli
-
expression of erythroid-specific isoform in MEL mutant under control of metallothionin promotor
-
expression of His-tagged wild-type, mutant K313A, mutant R149A and dimer mutant K313A/R149A, each subunit from 1 plasmid, in Escherichia coli hemA- mutant
-
overexpression of His-tagged wild-type and mutants in Escherichia coli
-
transient expression of erythroid-specific isoform and promotor-mutant in HeLa cells, luciferase reporter gene
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the enzyme is negatively regulated by heme at the level of mitochondrial import
in mouse model of erythropoietic protoporphyria, isoflurane induces 5-aminolevulinate synthase activity and increases excretion of porphyrin precursors
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
medicine
delivery of stable and highly active ALAS2 variants has the potential to expand and improve upon current photodynamic therapies regimes
medicine
-
in mouse model of erythropoietic protoporphyria, isoflurane induces 5-aminolevulinate synthase activity and increases excretion of porphyrin precursors. Mice homozygotically lacking ferrochelatase activity and receiving anaestesia show enhanced 5-aminolevulinate synthase and Cyp2E1 activities in the liver and increased urinary excretion of porphyrin precursors
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Ferreira, G.C.; Dailey, H.A.
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268
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Mus musculus
Hofer, T.; Wenger, R.H.; Kramer, M.F.; Ferreira, G.C.; Gassmann, M.
Hypoxic up-regulation of erythroid 5-aminolevulinate synthase
Blood
101
348-350
2003
Mus musculus
Lake-Bullock, H.; Dailey, H.A.
Biphasic ordered induction of heme synthesis in differentiating murine erythroleukemia cells: role of erythroid 5-aminolevulinate synthase
Mol. Cell. Biol.
13
7122-7132
1993
Mus musculus
Ferreira, G.C.; Vajapey, U.; Hafez, O.; Hunter, G.A.; Barber, M.J.
Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis
Protein Sci.
4
1001-1006
1995
Mus musculus
Ferreira, G.C.; Gong, J.
5-Aminolevulinate synthase and the first step of heme biosynthesis
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27
151-159
1995
Agrobacterium tumefaciens, Aspergillus nidulans, Bradyrhizobium japonicum, Saccharomyces cerevisiae, Gallus gallus, Euglena gracilis, Homo sapiens, Paracoccus denitrificans, Mus musculus, Rattus norvegicus, Sinorhizobium meliloti, Rhodobacter capsulatus, Cereibacter sphaeroides
Gong, J.; Kay, C.J.; Barber, M.J.; Ferreira, G.C.
Mutations at a glycine loop in aminolevulinate synthase affect pyridoxal phosphate cofactor binding and catalysis
Biochemistry
35
14109-14117
1996
Mus musculus, Rattus norvegicus
Tan, D.; Ferreira, G.C.
Active site of 5-aminolevulinate synthase resides at the subunit interface. Evidence from in vivo heterodimer formation
Biochemistry
35
8934-8941
1996
Saccharomyces cerevisiae, Mus musculus
Dailey, H.A.; Dailey, T.A.
Expression and purification of mammalian 5-aminolevulinate synthase
Methods Enzymol.
281
336-340
1997
Gallus gallus, Homo sapiens, Mus musculus, Rattus norvegicus
Tan, D.; Barber, M.J.; Ferreira, G.C.
The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase
Protein Sci.
7
1208-1213
1998
Mus musculus
Gong, J.; Hunter, G.A.; Ferreira, G.C.
Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor
Biochemistry
37
3509-3517
1998
Mus musculus
Tan, D.; Harrison, T.; Hunter, G.A.; Ferreira, G.C.
Role of arginine 439 in substrate binding of 5-aminolevulinate synthase
Biochemistry
37
1478-1484
1998
Mus musculus
Hunter, G.A.; Ferreira, G.C.
Lysine-313 of 5-aminolevulinate synthase acts as a general base during formation of the quinonoid reaction intermediates
Biochemistry
38
3711-3718
1999
Mus musculus
Zhang, J.; Ferreira, G.C.
Transient state kinetic investigation of 5-aminolevulinate synthase reaction mechanism
J. Biol. Chem.
277
44660-44669
2002
Mus musculus
Cheltsov, A.V.; Guida, W.C.; Ferreira, G.C.
Circular permutation of 5-aminolevulinate synthase: effect on folding, conformational stability, and structure
J. Biol. Chem.
278
27945-27955
2003
Mus musculus
Zhang, J.; Cheltsov, A.V.; Ferreira, G.C.
Conversion of 5-aminolevulinate synthase into a more active enzyme by linking the two subunits: spectroscopic and kinetic properties
Protein Sci.
14
1190-1200
2005
Mus musculus
Rodriguez, J.A.; Martinez, M.D.; Gerez, E.; Batlle, A.; Buzaleh, A.M.
Heme oxygenase, aminolevulinate synthetase and the antioxidant system in the brain of mice treated with porphyrinogenic drugs
Cell. Mol. Biol.
51
487-494
2005
Mus musculus
Hunter, G.A.; Zhang, J.; Ferreira, G.C.
Transient kinetic studies support refinements to the chemical and kinetic mechanisms of aminolevulinate synthase
J. Biol. Chem.
282
23025-23035
2007
Mus musculus
Buzaleh, A.M.; Moran-Jimenez, M.J.; Garcia-Bravo, M.; Sampedro, A.; Batlle, A.M.; Enriquez de Salamanca, R.; Fontanellas, A.
Induction of hepatic aminolevulinate acid synthetase activity by isoflurane in a genetic model for erythropoietic protoporphyria
Cell. Mol. Biol.
55
38-44
2009
Mus musculus
Okano, S.; Zhou, L.; Kusaka, T.; Shibata, K.; Shimizu, K.; Gao, X.; Kikuchi, Y.; Togashi, Y.; Hosoya, T.; Takahashi, S.; Nakajima, O.; Yamamoto, M.
Indispensable function for embryogenesis, expression and regulation of the nonspecific form of the 5-aminolevulinate synthase gene in mouse
Genes Cells
15
77-89
2010
Mus musculus
Lendrihas, T.; Hunter, G.A.; Ferreira, G.C.
Serine 254 enhances an induced fit mechanism in murine 5-aminolevulinate synthase
J. Biol. Chem.
285
3351-3359
2010
Mus musculus
Lendrihas, T.; Zhang, J.; Hunter, G.A.; Ferreira, G.C.
Arg-85 and Thr-430 in murine 5-aminolevulinate synthase coordinate acyl-CoA-binding and contribute to substrate specificity
Protein Sci.
18
1847-1859
2009
Mus musculus
Turbeville, T.D.; Zhang, J.; Adams, W.C.; Hunter, G.A.; Ferreira, G.C.
Functional asymmetry for the active sites of linked 5-aminolevulinate synthase and 8-amino-7-oxononanoate synthase
Arch. Biochem. Biophys.
511
107-117
2011
Mus musculus
Stojanovski, B.M.; Breydo, L.; Hunter, G.A.; Uversky, V.N.; Ferreira, G.C.
Catalytically active alkaline molten globular enzyme: effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase
Biochim. Biophys. Acta
1844
2145-2154
2014
Mus musculus (P08680)
Stojanovski, B.M.; Ferreira, G.C.
Murine erythroid 5-aminolevulinate synthase: adenosyl-binding site Lys221 modulates substrate binding and catalysis
FEBS open bio
5
824-831
2015
Mus musculus (P08680), Mus musculus
Stojanovski, B.M.; Hunter, G.A.; Jahn, M.; Jahn, D.; Ferreira, G.C.
Unstable reaction intermediates and hysteresis during the catalytic cycle of 5-aminolevulinate synthase: implications from using pseudo and alternate substrates and a promiscuous enzyme variant
J. Biol. Chem.
289
22915-22925
2014
Mus musculus (P08680), Mus musculus
Stojanovski, B.M.; Ferreira, G.C.
Asn-150 of murine erythroid 5-aminolevulinate synthase modulates the catalytic balance between the rates of the reversible reaction
J. Biol. Chem.
290
30750-30761
2015
Mus musculus (P08680), Mus musculus
Fratz, E.J.; Hunter, G.A.; Ferreira, G.C.
Expression of murine 5-aminolevulinate synthase variants causes protoporphyrin IX accumulation and light-induced mammalian cell death
PLoS ONE
9
e93078
2014
Mus musculus (P08680), Mus musculus
Stojanovski, B.M.; Breydo, L.; Uversky, V.N.; Ferreira, G.C.
The unfolding pathways of the native and molten globule states of 5-aminolevulinate synthase
Biochem. Biophys. Res. Commun.
480
321-327
2016
Mus musculus (P08680)
Stojanovski, B.M.; Breydo, L.; Uversky, V.N.; Ferreira, G.C.
Murine erythroid 5-aminolevulinate synthase Truncation of a disordered N-terminal extension is not detrimental for catalysis
Biochim. Biophys. Acta
1864
441-452
2016
Mus musculus (P08680), Mus musculus
Na, I.; Catena, D.; Kong, M.J.; Ferreira, G.C.; Uversky, V.N.
Anti-correlation between the dynamics of the active site loop and C-terminal tail in relation to the homodimer asymmetry of the mouse erythroid 5-aminolevulinate synthase
Int. J. Mol. Sci.
19
1899
2018
Mus musculus (P08680), Mus musculus
Na, I.; DeForte, S.; Stojanovski, B.M.; Ferreira, G.C.; Uversky, V.N.
Molecular dynamics analysis of the structural and dynamic properties of the functionally enhanced hepta-variant of mouse 5-aminolevulinate synthase
J. Biomol. Struct. Dyn.
36
152-165
2018
Mus musculus (P08680), Mus musculus, Rhodobacter capsulatus (P18079), Rhodobacter capsulatus, Rhodobacter capsulatus ATCC BAA-309 (P18079)
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