Information on EC 2.6.1.44 - alanine-glyoxylate transaminase and Organism(s) Homo sapiens and UniProt Accession P21549

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Homo sapiens
UNIPROT: P21549
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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria, Archaea


The taxonomic range for the selected organisms is: Homo sapiens

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EC NUMBER
COMMENTARY hide
2.6.1.44
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RECOMMENDED NAME
GeneOntology No.
alanine-glyoxylate transaminase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
L-alanine + glyoxylate = pyruvate + glycine
show the reaction diagram
pyridoxamine 5'-phosphate remains bound to the enzyme during the catalytic cycle. The enzyme-pyridoxamine 5'-phosphate complex displays a reactivity towards oxo acids higher than that of the apo-enzyme in presence of pyridoxamine
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
amino group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
glycine biosynthesis III
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glycine metabolism
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Alanine, aspartate and glutamate metabolism
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Glycine, serine and threonine metabolism
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Cysteine and methionine metabolism
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Metabolic pathways
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Biosynthesis of secondary metabolites
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SYSTEMATIC NAME
IUBMB Comments
L-alanine:glyoxylate aminotransferase
A pyridoxal-phosphate protein. With one component of the animal enzyme, 2-oxobutanoate can replace glyoxylate. A second component also catalyses the reaction of EC 2.6.1.51 serine---pyruvate transaminase.
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CAS REGISTRY NUMBER
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9015-67-2
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
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overexpression of human AGXT2 protects from asymmetric dimethylarginine-induced inhibition in nitric oxide production
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-alanine + glyoxylate
pyruvate + glycine
show the reaction diagram
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-
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?
glycine + pyruvate
L-alanine + glyoxylate
show the reaction diagram
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-
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?
L-alanine + glyoxylate
glycine + pyruvate
show the reaction diagram
transamination half-reaction kinetic parameters
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r
L-alanine + glyoxylate
pyruvate + glycine
show the reaction diagram
L-arginine + pyruvate
5-guanidino-2-oxopentanoate + L-alanine
show the reaction diagram
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-
-
?
L-asparagine + glyoxylate
4-amino-2,4-dioxobutanoate + glycine
show the reaction diagram
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-
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?
L-cysteine + pyruvate
3-mercapto-2-oxopropanoate + L-alanine
show the reaction diagram
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enzyme catalyzes both beta-elimination and half-transamination of L-cysteine together with pyruvate transamination via a ketimine common intermediate. L-cysteine partitions between the two reactions with a ratio of 2.5
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?
L-glutamate + glyoxylate
2-oxoglutaramate + glycine
show the reaction diagram
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-
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?
L-phenylalanine + glyoxylate
phenylpyruvate + glycine
show the reaction diagram
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-
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?
L-serine + glyoxylate
3-hydroxy-2-oxopropanoate + glycine
show the reaction diagram
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-
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?
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
L-alanine + glyoxylate
pyruvate + glycine
show the reaction diagram
additional information
?
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A2V838
enzyme is highly specific for catalyzing glyoxylate to glycine processing, playing a key role in glyoxylate detoxification
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
pyridoxal 5'-phosphate
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pyridoxal 5'-phosphate
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
amino-oxyacetic acid
aminooxyacetic acid
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D-alanine
pyruvate
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
aminooxyacetic acid
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.038
glyoxylate
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pH 7.4, 25°C
52
L-alanine
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pH 7.4, 25°C
22
glycine
0.13 - 2.5
glyoxylate
9.1 - 46
L-alanine
1
L-cysteine
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pH 7.4, Km value of L-cysteine is decreased by 40fold and 200fold in comparison with those of L-alanine and L-serine
0.21
pyruvate
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.147
L-alanine
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pH 7.4, 25°C
0.33
glycine
0.068 - 45
glyoxylate
0.07 - 45
L-alanine
0.22
L-cysteine
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pH 7.4
0.36
pyruvate
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.45 - 196
glyoxylate
0.005 - 1.4
L-alanine
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
14.3 - 28.7
D-alanine
2.3 - 22.8
pyruvate
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
51.8
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purified recombinant enzyme
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 8.5
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recombinant enzyme
7.8 - 8
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 8.2
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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expressed primarily in the kidney
Manually annotated by BRENDA team
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LOCALIZATION
ORGANISM
UNIPROT
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GeneOntology No.
LITERATURE
SOURCE
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42700
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about 42700 Da, SDS-PAGE
48600
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SDS-PAGE
52000
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FLAG-tagged enzyme, SDS-PAGE
90000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 43000, SDS-PAGE
homodimer
additional information
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant S187F, to a resolution of 2.9 A. The overall conformation of the variant is similar to that of normal AGTwith a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This results in a mispositioning of the AGT-pyridoxamine 5'-phosphate complex and of the external aldimine
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crystals belong to space group P4(1)2(1)2 or its enantiomorph with uni-cell parameters a = b = 90.81, c = 142.62 A
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using 10% (w/v) PEG 4000 in 0.1 M Na HEPES pH 7.5
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70.1
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the melting temperature of the minor allele holoenzyme is at 70.1°C
76.3
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the melting temperature of the major allele holoenzyme is at 76.3°C
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
human AGT can substitute for function of yeast Agx1 (Yeast alanine:glyoxylate aminotransferase) and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast
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major allele (P11/I340) is more stable against increasing urea concentrations than minor allele (P11L/I340M) or mutant protein P11L/I340M/G170R
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partial digestion by trypsin provides an indicator of proper folding of the enzyme, while for some mutants, sensitivity to trypsin can be ameliorated by addition of pyridoxal 5'-phosphate or aminooxyacetic acid
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partial trypsin digestion provides an indicator of proper folding of the mutant enzyme. For selected mutations the sensitivity to trypsin can be ameliorated by addition of pyridoxal phosphate or aminooxy acetic acid as specific pharmacological chaperones
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, wild-type and mutant enzyme G82E are stable for at least 1 month
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nickel-resin affinity chromatography, gel filtration
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recombinant wild-type and G82E mutant His-tagged enzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
AGXT cDNA cloned, AGXT*LTM expressed as a GST-fusion protein in Escherichia coli BL21(RIL) and in Sf9 cells
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cloned and expressed in Escherichia coli JM109
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expressed in COS-1a cells
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expressed in COS-7 cells and human umbilical vein endothelial cells with a C-terminal FLAG epitope tag
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expressed in Escherichia coli
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expressed in Escherichia coli JM109 cells
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expressed in Mus musculus embryonic stem cells
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expressed in stably transformed CHO cells
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expression in CHO cell
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expression in Escherichia coli
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expression in HeLa cell; expression vectors of the enhanced green fluorescent protein-tagged alanine:glyoxylate aminotransferase and deletion mutants are introduced into HeLa cells to identify the peroxisomal targeting signal of the alanine:glyoxylate aminotransferase
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expression of of untagged alanine-glyoxylate aminotransferase in Escherichia coli
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for sequence determination, GFP-fusion proteins used in localization experiments
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His-tagged protein expressed in Escherichia coli JM109, expressed in CHO cells
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human AGT can substitute for function of yeast Agx1 (yeast alanine:glyoxylate aminotransferase) and that mutations associated with disease in humans show reduced growth in yeast. The reduced growth of minor allele mutants reflects reduced protein levels, indicating that these proteins are less stable than wild-type AGT in yeast
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human AGT expressed in Escherichia coli B834(DE3)
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mammalian expression vector pHYK, expressed in COS-1 cells
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wild-type and G82E mutant His-tagged enzyme expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
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LITERATURE
A280V
natural mutant from patient with primary hyperoxaluria type 1, 92% of normal enzyme activity
G161R
natural mutant from patient with primary hyperoxaluria type 1, 6.2% of normal enzyme activity
I279T
natural mutant from patient with primary hyperoxaluria type 1, 98% of normal enzyme activity
S187F
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mutation gives rise to a variant associated with primary hyperoxaluria type I. Mutation shows a 300- to 500fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, a different pyridoxamine 5'-phosphate binding mode and affinity, and a different microenvironment of the external aldimine
S218L
natural mutant from patient with primary hyperoxaluria type 1, 10% of normal enzyme activity
A112D
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less than 5% of the specific activity of the wild type enzyme
C173Y
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less than 5% of the specific activity of the wild type enzyme
D183N
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less than 5% of the specific activity of the wild type enzyme
DELTA 1-21
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purified protein does not show bound PLP (affinity is about 80fold lower than wild type protein), catalytic activity about 1000fold lower than wild type protein, expressed in Escherichia coli in an insoluble form, peroxisomal localization, expressed in CHO cells the mutant protein forms large stable but catalytically inactive aggregates in the peroxisomes
F152A
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the mutant shows decreased activity compared to the wild type enzyme
F52I
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natural mutation in enzyme major allele, 13% of the activity of major allele; natural mutation in enzyme minor allele, 14% of the activity of minor allele
G156R
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less than 5% of the specific activity of the wild type enzyme
G161C
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5% of wild-type expression level, reduced catalytic activity
G161S
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12% of wild-type expression level, reduced catalytic activity
I244T
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natural mutation in enzyme minor allele, 8-26% of the activity of major allele, in vitro
I340M
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polymorphism associated with enzyme from minor allele, significantly higher Km-value than that for major allele, 90% of activity of enzyme from major allele
K209R
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less than 5% of the specific activity of the wild type enzyme
P10L/P11L
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Kcat value 56% of wild type protein, aggregation occuring at a slower rate than that of DELTA 1-21 protein
P11L/F152I/I340M
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naturally occuring mutations, mistargeted to the mitochondria, forms dimers, catlytically active
P11L/G170R/I340M
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naturally occuring mutations, creates a hidden N-terminal mitochondrial targeting sequence, the unmasking of which occurs in the hereditary calcium oxalate kidney stone disease primary hyperoxaluria type 1; this unmasking is due to the additional presence of a common disease-specific G170R mutation, forms dimers, catalytically active
P11L/G41R/I340M
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naturally occuring mutations, mistargeted to the mitochondria, catalytically inactive, aggregates
P11L/I244T/I340M
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naturally occuring mutations, mistargeted to the mitochondria, forms dimers, catlytically active
P11L/I340M
P11L/I340M/F152I
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naturally occuring mutation, possibly mistargeting into mitochondrial matrix
P11L/I340M/G170R
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naturally occuring mutations, pathogenic variant
P11L/I340M/G41R
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naturally occuring mutation, predicted to be responsible for the depletion of immunoreactive enzyme protein and formation of intraperoxisomal aggregates
S158L
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natural mutation in enzyme major allele, no in vitro enzymic activity
S187F
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less than 5% of the specific activity of the wild type enzyme
S218L
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less than 5% of the specific activity of the wild type enzyme
V336D
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natural mutation in enzyme major allele, 22.4% of the activity of major allele; natural mutation in enzyme minor allele, 5.2% of the activity of minor allele
W108R
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less than 5% of the specific activity of the wild type enzyme
W251K
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naturally occuring mutation, mutant protein localized in peroxisome and cytosol
additional information
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Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
denaturation of enzyme with guanidine-HCl and re-folding, complete renaturation. Mutations G41V and G41R, associated with primary hyperoxaluria type I, show enhanced activity after re-folding. Pyridoxal 5’-phosphate is not required for proper re-folding
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quick dilution (100fold) to an urea concentration that would be expected to support native enzyme, only about 20 and 5% of activity is recovered for major allele (P11/I340) and P11L/I340M (minor allele, respectively)
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
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development of an indirect glycolate cytotoxicity assay using CHO cells expressing glycolate oxidase and various normal and mutant forms of AGT
medicine
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LINKS TO OTHER DATABASES (specific for EC-Number 2.6.1.44)
ExplorEnz
ExPASy
KEGG
MetaCyc
SABIO-RK
NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM
IUBMB Enzyme Nomenclature
PROSITE Database of protein families and domains
InterPro (database of protein families, domains and functional sites)