Information on EC 2.4.2.14 - amidophosphoribosyltransferase

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

EC NUMBER
COMMENTARY
2.4.2.14
-
RECOMMENDED NAME
GeneOntology No.
amidophosphoribosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate = L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
show the reaction diagram
mechanism of glutamine amide transfer
-
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate = L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
show the reaction diagram
binding of 5-phospho-alpha-D-ribose 1-diphosphate activates the enzyme by a structural change that lowers the Km for glutamine 100fold and couples glutamine hydrolysis to synthesis of 5-phospho-beta-D-ribosylamine
-
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate = L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
amino group transfer
-
-
-
-
amino group transfer
P0AG16
first step of de novo purine nucleotide biosynthesis
amino group transfer
Q9SI61, Q9STG9, Q9T0J5
; first step of de novo purine biosynthesis; first step of de novo purine biosynthesis
PATHWAY
KEGG Link
MetaCyc Link
4-amino-2-methyl-5-phosphomethylpyrimidine biosynthesis (yeast)
-
5-aminoimidazole ribonucleotide biosynthesis I
-
5-aminoimidazole ribonucleotide biosynthesis II
-
Alanine, aspartate and glutamate metabolism
-
Biosynthesis of secondary metabolites
-
Metabolic pathways
-
Purine metabolism
-
superpathway of 5-aminoimidazole ribonucleotide biosynthesis
-
SYSTEMATIC NAME
IUBMB Comments
5-phospho-beta-D-ribosylamine:diphosphate phospho-alpha-D-ribosyltransferase (glutamate-amidating)
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5'-phosphoribosylpyrophosphate amidotransferase
-
-
-
-
5-phosphoribosyl-1-pyrophosphate amidotransferase
-
-
-
-
5-phosphoribosylpyrophosphate amidotransferase
-
-
-
-
5-phosphororibosyl-1-pyrophosphate amidotransferase
-
-
-
-
alpha-5-phosphoribosyl-1-pyrophosphate amidotransferase
-
-
-
-
amidotransferase, phosphoribosyl pyrophosphate
-
-
-
-
ATASE
-
-
-
-
AtGPRT
Q9SI61, Q9STG9, Q9T0J5
-
Gln phosphoribosylpyrophosphate amidotransferase
Q9SI61, Q9STG9, Q9T0J5
-
glutamine 5-phosphoribosylpyrophosphate amidotransferase
-
-
-
-
glutamine 5-phosphoribosylpyrophosphate amidotransferase
-
-
glutamine phosphoribosylpyrophosphate amidotransferase
-
-
-
-
glutamine phosphoribosylpyrophosphate amidotransferase
Q9SI61, Q9STG9, Q9T0J5
-
glutamine phosphoribosylpyrophosphate amidotransferase
-
-
glutamine ribosylpyrophosphate 5-phosphate amidotransferase
-
-
-
-
GPAT
-
-
-
-
GPATase
-
-
-
-
GPATase
-
-
GPRAT
Q9SI61, Q9STG9, Q9T0J5
-
phosphoribose pyrophosphate amidotransferase
-
-
-
-
phosphoribosyl pyrophosphate amidotransferase
-
-
-
-
phosphoribosylamidotransferase
F2XMV3
-
phosphoribosylamidotransferase
Coniothyrium minitans ZS-1
F2XMV3
-
-
phosphoribosyldiphosphate 5-amidotransferase
-
-
-
-
phosphoribosylpyrophosphate glutamyl amidotransferase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9031-82-7
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
isoform AtGPRAT1
UniProt
Manually annotated by BRENDA team
isoform AtGPRAT2, At4g34740
UniProt
Manually annotated by BRENDA team
isoform AtGPRAT3
UniProt
Manually annotated by BRENDA team
brine shrimp, activity increases 7fold during early larval development in 2 d old Artemia
-
-
Manually annotated by BRENDA team
pigeon
-
-
Manually annotated by BRENDA team
Coniothyrium minitans ZS-1
-
UniProt
Manually annotated by BRENDA team
strains K-12 or B-96, purine requiring strain
-
-
Manually annotated by BRENDA team
soybean, Merr. cv. Williams
-
-
Manually annotated by BRENDA team
no activity in Escherichia coli
strain DM1293, lacking purF gene product, purF77::TN10
-
-
Manually annotated by BRENDA team
no activity in Escherichia coli DM1293
strain DM1293, lacking purF gene product, purF77::TN10
-
-
Manually annotated by BRENDA team
no activity in Salmonella sp.
Salmonella enterica strain DM1936, lacking purF gene product, purF2085
-
-
Manually annotated by BRENDA team
wild type strain 972h- and mutant strain ade 2h- devoid of adenylosuccinate synthase
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-, F2XMV3
essential for conidiation
physiological function
Coniothyrium minitans ZS-1
-
essential for conidiation
-
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
ir
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
-
-
ir
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
reaction at 50% the rate of aminotransferase activity
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
no activity with carbamoyl phosphate
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
glutamine binding site distinct from NH3-site
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
reaction at 33% the rate of amminotransferase activity
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + L-glutamate + diphosphate
show the reaction diagram
-
reaction at 70% the rate of aminotransferase activity
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
rate-limiting enzyme of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
regulating enzyme of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
enzyme of purine biosynthetic pathway, regulatory enzyme in the flow of recently fixed nitrogen from initial assimilation into amino acids via purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
P0AG16
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
Q9SI61, Q9STG9, Q9T0J5
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
-
in Salmonella enterica, the biosynthetic pathways for the generation of purines and the essential cofactor thiamine pyrophosphate branch after sharing five enzymatic steps. Phosphoribosyl amine is the first intermediate in the common portion of the pathway and is generated from phosphoribosylpyrophosphate and glutamine by the PurF enzyme (phosphoribosylpyrophosphate amidotransferase). Tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase, composed of the TrpD and TrpE proteins, is essential for phosphoribosyl amine formation in strains lacking both yjgF and purF
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
Q9SI61, Q9STG9, Q9T0J5
first step of de novo purine biosynthesis
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
Q9SI61, Q9STG9, Q9T0J5
40 mM glutamine, 5 mM PRPP, 10 mM dithiothreitol
glutamate dehydrogenase-coupled assay, glutamate-dependent reduction of 1.36 mM 3-acetylpyridine dinucleotide measurable as increase of absorbance at 363 nm
-
-
L-glutamine + H2O
L-glutamate + NH3
show the reaction diagram
-
-
-
-
L-glutamine + H2O
L-glutamate + NH3
show the reaction diagram
-
-
-
-
NH3 + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
-
-
?
NH3 + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
-
-
?
NH3 + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
no activity with NH4+
-
?
NH3 + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
2.8fold higher aminotransferase activity compared to amidotransferase activity
-
?
NH3 + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
2fold higher aminotransferase activity compared to amidotransferase activity
-
?
NH3 + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
3fold higher aminotransferase activity compared to amidotransferase activity
-
?
NH3 + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
lower aminotransferase activity compared to amidotransferase activity
-
?
NH3 + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
NH3-binding site is distinct from glutamine-site
-
?
phosphoribosylanthranilate
indoleglycerol phosphate + CO2 + H2O
show the reaction diagram
P0AG16
5 M PurF mutant I198V or variant 1-04, in presence of 0.5 mM beta-mercaptoethanol, pH 8.6, 23 C, 16 h
Amadori rearrangement of aminoaldose (phosphoribosylanthranilate) to aminoketose (indoleglycerol phosphate)
-
?
L-glutamine + H2O
L-glutamate + NH3
show the reaction diagram
-
enzyme exhibits glutaminase activity in the absence of other substrates or effectors
-
?
additional information
?
-
Q9SI61, Q9STG9, Q9T0J5
no GPRAT activity detectable in cell extracts of recombinant AtGPRAT1 expressing Escherichia coli
-
-
-
additional information
?
-
P0AG16
GPATase-thioester-5'-phosphoribosylpyrophosphate complex model demonstrates the ammonia transfer between the active sites of GPATase in its active conformation. The ammonia channel in GPATase is a transient structural element, a pipe through which ammonia travels in the absence of an external driving potential. The ammonia tunnel in GPATase discriminates against ammonium ion
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
first reaction in de-novo pathway of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
rate-limiting enzyme of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
regulating enzyme of purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
L-glutamate + 5-phospho-beta-D-ribosylamine + diphosphate
show the reaction diagram
-
enzyme of purine biosynthetic pathway, regulatory enzyme in the flow of recently fixed nitrogen from initial assimilation into amino acids via purine biosynthesis
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
P0AG16
-
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
Q9SI61, Q9STG9, Q9T0J5
-
-
-
-
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
-
in Salmonella enterica, the biosynthetic pathways for the generation of purines and the essential cofactor thiamine pyrophosphate branch after sharing five enzymatic steps. Phosphoribosyl amine is the first intermediate in the common portion of the pathway and is generated from phosphoribosylpyrophosphate and glutamine by the PurF enzyme (phosphoribosylpyrophosphate amidotransferase). Tryptophan biosynthetic enzyme complex anthranilate synthase-phosphoribosyltransferase, composed of the TrpD and TrpE proteins, is essential for phosphoribosyl amine formation in strains lacking both yjgF and purF
-
-
?
L-glutamine + 5-phospho-alpha-D-ribose 1-diphosphate + H2O
5-phospho-beta-D-ribosylamine + diphosphate + L-glutamate
show the reaction diagram
Q9SI61, Q9STG9, Q9T0J5
first step of de novo purine biosynthesis
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Ca2+
-
activation, approx. 15% as effective as Mg2+ or Mn2+
Co2+
-
activation, approx. 40% as effective as Mg2+ or Mn2+
Fe
-
enzyme contains a [4Fe-4S] cluster; iron-sulfur center can be removed with 1,10-phenanthroline, resulting apoprotein is devoid of amino- and amidotransferase activity; required, iron-sulfur protein
Fe
-
enzyme contains a [4Fe-4S] cluster; iron is oxidized by O2 to enzyme-bound Fe3+
Fe
-
enzyme contains a [4Fe-4S] cluster
Fe
-
enzyme contains a [4Fe-4S] cluster
Fe
-
enzyme contains a [4Fe-4S] cluster; loss of activity after oxidation of the iron-sulfur center
Fe
-
enzyme contains a diamagnetic [4Fe-4S] cluster essential for activity
Fe
-
low temperature magnetic circular dichroism, electron paramagnetic resonance and resonance Raman spectroscopy of oxidized and reduced [4Fe-4S] cluster, native enzyme contains an [4Fe-4S]2+ cluster
Mg2+
-
required
Mg2+
-
maximal activation at concentrations 2.5-5 times higher than that of the corresponding 5-phospho-alpha-D-ribose 1-diphosphate concentration; required
Mg2+
-
Km-value: 0.65 mM, inhibition above 10 mM; required
Mg2+
-
5-phospho-alpha-D-ribose 1-diphosphate-Mg3- is the reactive molecular species; required
Mn2+
-
required, equally effective as Mg2+
additional information
-
non-heme iron is not present in significant amounts; not activated by iron or sulfide
additional information
-
4Fe-4S-cluster; sulfur required, iron-sulfur protein
additional information
-
4Fe-4S-cluster; S2- is oxidized by O2 to a mixture of sulfur oxides bound as thiocysteine and yet unidentified products
additional information
-
4Fe-4S-cluster
additional information
-
-
additional information
-
4Fe-4S-cluster
additional information
-
probably diamagnetic
additional information
-
non-heme iron is not present in significant amounts
additional information
-
-
additional information
-
not activated by Ba2+, Cd2+, Cu2+, Fe2+, Hg2+, Ni2+, Zn2+
additional information
-
low temperature magnetic circular dichroism, electron paramagnetic resonance and resonance Raman spectroscopy
additional information
-
-
additional information
-
prototype for a metal-free amidophosphoribosyltransferase
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1-alpha-diphosphoryl-2-alpha,3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5-phosphate
-
competitive vs. 5-phospho-alpha-D-ribose 1-diphosphate in wild-type and P410W mutant, competition for the amidotransferase C site
2-amino-4-oxo-5-chloropentanoate
-
approx. 80% inactivation of NH3-dependent activity after 30 min
5',5'''-P1,P4-Diguanosine tetraphosphate
-
IC50: 0.4 mM, 2 mM, approx. 80% inhibition
5'-p-fluorosulfonylbenzoyladenosine
-
inactivation follows pseudo-first order kinetic, AMP, GMP and 5-phospho-alpha-D-ribose 1-diphosphate protect
6-diazo-5-oxo-L-norleucine
-
approx. 98% inactivation of amidotransferase activity after 30 min
6-diazo-5-oxo-L-norleucine
-
competitive vs. glutamine, inactivation half-life: 11 min
6-iodopurine
-
5 mM, 39% inhibition
6-mercaptopurine ribonucleotide
-
5 mM, 64% inhibition
adenine nucleotide
Q9SI61, Q9STG9, Q9T0J5
maximal inhibition by combination of adenine nucleotides (in total 10 mM), inhibitory effect of single nucleotides (5 mM) increases in the order AMP, ATP, ADP
ADP
-
1 mM, approx. 95% inhibition
ADP
-
5 mM, 40% inhibition
ADP
P00497
50% inhibition of wild-type, S283A, K305Q, R307Q and S347A mutant enzyme at 4.7 mM, 24 mM, 31 mM, 28 mM and 8.1 mM respectively
ADP
-
4.1 mM, 50% inhibition
allopurinol ribonucleotide
-
5 mM, 68% inhibition
AMP
-
approx. 10 mM, complete inhibition, sigmoidal inhibition curve, competitive vs. 5-phospho-alpha-D-ribose 1-diphosphate, GMP and AMP together have a synergistic effect on inhibition
AMP
-
1 mM, approx. 40% inhibition
AMP
-
no inhibition in the presence of 5-phospho-alpha-D-ribose 1-diphosphate at high concentrations
AMP
-
1.8 mM, 50% inhibition
AMP
-
5 mM, 76% inhibition, noncompetive vs. glutamine
AMP
-
5 mM, 79% and 24% inhibition of aminotransferase and amidotransferase activity at 1 mM 5-phospho-alpha-D-ribose 1-diphosphate
AMP
-
competitive vs. 5-phospho-alpha-D-ribose 1-diphosphate
AMP
-
2.7 mM and 1.2 mM, 50% inhibition of stable and unstable enzyme preparation respectively
AMP
-
5 mM, 50% inhibition
AMP
-
4.7 mM, 50% inhibition
AMP
-
competitive vs. 5-phospho-alpha-D-ribose 1-diphosphate, noncompetitive vs. glutamine
AMP
P00497
50% inhibition of wild-type, S283A, K305Q, R307Q and S347A mutant enzyme at 0.9 mM, 6.1 mM, 2.5 mM, 2.6 mM and 1.5 mM respectively
AMP
-
6.12 mM, 50% inhibition
ATP
-
5 mM, 21% inhibition
ATP
-
10 mM, 10% residual activity
azaserine
-
competitive vs. glutamine, inactivation half-life: 12 min
CMP
-
5 mM, 22% inhibition
DAS734
Q9SI61, Q9STG9, Q9T0J5
phenyltriazole acetic acid herbicide, [5-(4-chlorophenyl)-1-isopropyl-1H-[1,2,4]triazol-3-yl]-acetic acid, inhibits Arabidopsis root growth by 50% at 200 nM, phytotoxicity can be alleviated by addition of adenine, no inhibitory effect on Escherichia coli; phenyltriazole acetic acid herbicide, [5-(4-chlorophenyl)-1-isopropyl-1H-[1,2,4]triazol-3-yl]-acetic acid, non-competitive with respect to L-glutamine with slow, tight-binding behavior, inhibits Arabidopsis root growth by 50% at 200 nM, phytotoxicity can be alleviated by addition of adenine, no inhibitory effect on Escherichia coli
dCMP
-
5 mM, 26% inhibition
diphosphate
-
uncompetitive vs. 5-phospho-alpha-D-ribose 1-diphosphate
GDP
-
1 mM, approx. 75% inhibition
GDP
-
5 mM, 40% inhibition
GDP
-
8.5 mM, 50% inhibition
glutamate
-
competitive vs. glutamate
GMP
-
approx. 2.5 mM, complete inhibition, highly sigmoidal inhibition curve, competitive vs. 5-phospho-alpha-D-ribose 1-diphosphate, GMP and AMP together have an synergistic effect on inhibition
GMP
-
1 mM, approx. 60% inhibition
GMP
-
no inhibition in the presence of 5-phospho-alpha-D-ribose 1-diphosphate at high concentrations
GMP
-
0.5 mM; 50% inhibition
GMP
-
0.5 mM; 5 mM, 70% inhibition
GMP
-
0.5 mM; 5 mM, 56% and 12% inhibition of aminotransferase and amidotransferase activity at 1 mM 5-phospho-alpha-D-ribose 1-diphosphate
GMP
-
0.44 mM and 0.25 mM, 50% inhibition of stable and unstable enzyme preparation respectively
GMP
-
2 mM, 50% inhibition
GMP
-
1.2-1.4 mM, 50% inhibition
GMP
P00497
50% inhibition of wild-type, S283A, K305Q, R307Q and S347A mutant enzyme at 9.4 mM, 6.6 mM, 50 mM, 50 mM and 145 mM respectively
GMP
-
IC50: 7.9 mM
GTP
-
5 mM, 79% inhibition
GTP
-
5 mM, 13% inhibition
GTP
-
10 mM, 10% residual activity
IDP
-
5 mM, 29% inhibition
IMP
-
5 mM, 86% inhibition
IMP
-
5 mM, 41% inhibition
IMP
P00497
50% inhibition of wild-type and S347A mutant enzyme at 26 mM and 41 mM respectively
iodoacetamide
-
approx. 60% inactivation of amidotransferase activity after 30 min, very weak inactivation of NH3-dependent activity
N3-AMP
-
1.2-1.4 mM, 50% inhibition, AMP and GMP protect
NH3
-
inhibition of amidotransferase activity
NH4+
-
competitive vs. glutamine
OMP
-
5 mM, 25% inhibition
p-mercuribenzoate
-
0.001 mM, complete inhibition
phosphate
-
competitive vs. 5-phospho-alpha-D-ribose 1-diphosphate
phosphate
-
20 mM, 50% inhibition
piritrexim
-
noncompetitive vs. 5-phospho-alpha-D-ribose 1-diphosphate, binds with positive cooperativity at 2 allosteric sites of an inactive dimer
purine nucleotide
Q9SI61, Q9STG9, Q9T0J5
allosteric feedback inhibition by end products of the purine biosynthesis pathway, act on the PRPP domain
-
TMP
-
5 mM, 42% inhibition
TTP
-
5 mM, 12% inhibition
UDP
-
5 mM, 19% inhibition
UMP
-
5 mM, 25% inhibition
UTP
-
5 mM, 16% inhibition
XDP
-
5 mM, 27% inhibition
XMP
-
51% inhibition; 5 mM
XMP
-
5 mM, 41% inhibition
XMP
-
competitive vs. 5-phospho-alpha-D-ribose 1-diphosphate
XTP
-
5 mM, 44% inhibition
methyl-dCMP
-
5 mM, 47% inhibition
additional information
-
not inhibited by ribose 5-phosphate, purine ribonucleosides or bases, 2'- or 3'-phosphate or deoxyribose phosphate analogues or by pyrimidine ribonucleotides
-
additional information
-
synergistic inhibition of glutaminase activity by AMP plus GMP and N3-AMP plus GMP
-
additional information
-
synergistic inhibition by AMP and GMP, binding of GMP to an allosteric i.e. A site and AMP to a proximal catalytic i. e. C site are necessary for synergistic inhibition
-
additional information
P00497
strong synergistic inhibition with ADP and GMP
-
additional information
Q9SI61, Q9STG9, Q9T0J5
minimal inhibition by inosine monophosphate (IMP) and guanosine monophosphate (GMP)
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Mg-phosphoribosyldiphosphate
-
no glutamine-binding site in the absence of Mg-phosphoribosyldiphosphate
-
Mg-phosphoribosyldiphosphate
-
-
-
phosphoribosyl-5-phosphate
-
3-4fold activation of glutaminase activity in the presence of phosphate or diphosphate
Mg-phosphoribosyldiphosphate
-
requirement, phosphoribosyldiphosphate-Mg3- is the reactive molecular species of phosphoribosyldiphosphate, Km-value: 0.62 mM
-
additional information
-
complex allosteric enzyme whose activity is regulated by a series of conformational changes induced by a number of ligands
-
additional information
-
-
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.031
-
5-phospho-alpha-D-ribose 1-diphosphate
-
P410W mutant enzyme
0.053
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.067
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.072
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.086
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.14
0.48
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.24
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.4
-
5-phospho-alpha-D-ribose 1-diphosphate
-
cosubstrate NH3
0.4
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.47
-
5-phospho-alpha-D-ribose 1-diphosphate
-
adenocarcinoma 755
0.48
-
5-phospho-alpha-D-ribose 1-diphosphate
-
in Tris buffer
0.57
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.66
-
5-phospho-alpha-D-ribose 1-diphosphate
-
-
0.7
-
5-phospho-alpha-D-ribose 1-diphosphate
-
cosubstrate glutamine
0.87
-
5-phospho-alpha-D-ribose 1-diphosphate
-
at 90C
193
-
glutamine
-
5-phospho-alpha-D-ribose 1-diphosphate-independent glutamine hydrolysis
0.1
-
L-glutamine
-
-
0.53
-
L-glutamine
-
-
0.64
-
L-glutamine
-
N101G mutant enzyme, 5-phospho-alpha-D-ribose 1-diphosphate-dependent glutaminase activity
0.75
-
L-glutamine
-
-
1
4.5
L-glutamine
-
-
1.24
1.5
L-glutamine
-
-
1.34
-
L-glutamine
Q9SI61, Q9STG9, Q9T0J5
cell extracts of recombinant AtGPRAT2 expressing Escherichia coli
1.42
-
L-glutamine
-
N101D mutant enzyme, 5-phospho-alpha-D-ribose 1-diphosphate-dependent glutaminase activity
1.6
-
L-glutamine
-
-
1.7
-
L-glutamine
-
-
1.72
-
L-glutamine
-
5-phospho-alpha-D-ribose 1-diphosphate-dependent glutamine hydrolysis; wild-type enzyme, 5-phospho-alpha-D-ribose 1-diphosphate-dependent glutaminase activity
1.8
-
L-glutamine
-
adenocarcinoma 755
2.1
-
L-glutamine
-
-
2.43
-
L-glutamine
-
G102A mutant enzyme, 5-phospho-alpha-D-ribose 1-diphosphate-dependent glutaminase activity
3
-
L-glutamine
-
at 90C
4.3
-
L-glutamine
-
-
6.03
-
L-glutamine
-
N101G mutant enzyme, aminotransferase activity
6.08
-
L-glutamine
-
D127A mutant enzyme, aminotransferase activity
7.31
-
L-glutamine
-
R73L mutant enzyme, aminotransferase activity
7.34
-
L-glutamine
-
wild-type enzyme, aminotransferase activity
7.67
-
L-glutamine
-
G102A mutant enzyme, aminotransferase activity
9.17
-
L-glutamine
-
N101D mutant enzyme, aminotransferase activity
9.76
-
L-glutamine
-
R73H mutant enzyme, aminotransferase activity
18
-
L-glutamine
-
-
101
-
L-glutamine
-
R73H mutant enzyme, 5-phospho-alpha-D-ribose 1-diphosphate-dependent glutaminase activity
110
-
L-glutamine
-
R73L mutant enzyme, 5-phospho-alpha-D-ribose 1-diphosphate-dependent glutaminase activity
236
-
L-glutamine
-
D127A mutant enzyme, 5-phospho-alpha-D-ribose 1-diphosphate-dependent glutaminase activity
16
-
NH3
-
-
0.65
-
Mg2+
-
-
additional information
-
additional information
-
kinetic study
-
additional information
-
additional information
-
effect of AMP on kinetic parameters
-
additional information
-
additional information
-
kinetic properties of amido- and aminotransferase activity
-
additional information
-
additional information
-
kinetic study
-
additional information
-
additional information
-
kinetic constants of Arg73 and Tyr74 mutants for basal and total glutaminase activity
-
16
-
NH4+
-
-
additional information
-
phosphoribosylanthranilate
P0AG16
variant 1-04, kcat/Km = 0.3 1/s*M, initial velocity measurements over time at substrate concentrations to up to 2 mM
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
additional information
-
phosphoribosylanthranilate
P0AG16
mutant I198V, modestly higher rate than spontaneous substrate hydrolysis of <1/h; variant 1-04, 25 to 30-fold more active than mutant I198V but 2.3*10(7)-fold less efficient than phosphoribosylanthranilate isomerase; variant 1-04, conversion of 5.5% of available substrate, 22 turnovers per active site
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.053
-
1-alpha-diphosphoryl-2-alpha,3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5-phosphate
-
P410W mutant enzyme
0.058
-
1-alpha-diphosphoryl-2-alpha,3-alpha-dihydroxy-4-beta-cyclopentane-methanol-5-phosphate
-
-
0.39
-
5'-p-fluorosulfonylbenzoyladenosine
-
-
0.0034
-
6-diazo-5-oxo-L-norleucine
-
-
0.038
0.64
ADP
-
-
0.04
-
AMP
-
-
0.092
2.5
AMP
-
-
0.031
1.1
ATP
-
-
4
-
azaserine
-
-
0.65
-
diphosphate
-
-
0.38
5.4
GDP
-
-
30
-
glutamate
-
-
0.086
0.35
GMP
-
-
0.18
3.5
IMP
-
-
3.3
-
NH3
-
-
16
-
NH4+
-
-
0.066
-
piritrexim
-
-
1.2
-
XMP
-
-
IC50 VALUE [mM]
IC50 VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.0002
-
DAS734
Q9SI61, Q9STG9, Q9T0J5
10 min preincubation of recombinant enzyme with inhibitor leads to maximum inhibition, slow but reversible binding; cell extracts of recombinant AtGPRAT3 expressing Escherichia coli
7.9
-
GMP
-
IC50: 7.9 mM
0.4
-
5',5'''-P1,P4-Diguanosine tetraphosphate
-
IC50: 0.4 mM, 2 mM, approx. 80% inhibition
additional information
-
DAS734
Q9SI61, Q9STG9, Q9T0J5
200 nM leads to 50% inhibition of Arabidopsis root growth (RI50) in wild-type AtGPRAT2 expressing seedlings; 20-35 microM leads to 50% inhibition of Arabidopsis root growth (RI50) at in presence of endogenous AtGPRAT2 harbouring R264K polymorphism; >500-fold higher for mutant R264K compared to wild-type
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.001
-
-
L-glutamine
0.00125
0.0014
-
-
0.00297
-
-
NH3
0.0055
-
-
-
3
-
-
wild-type, pH 8.0, 37C
15.6
-
-
-
additional information
-
-
assay method to measure both amidotransferase and aminotransferase activity
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6
8
-
broad, phosphate buffer
6.5
8.5
-
broad, Tris or phosphate buffer, constant activity
6.5
8.5
-
glutaminase activity
6.5
-
-
imidazole/HCl buffer
6.6
7.7
-
broad, amidotransferase activity, phosphate buffer
6.8
7.4
-
Tris/HCl buffer preferred
7.5
-
-
Tris/HCl buffer
7.5
-
-
unstable enzyme
7.8
8.6
-
broad, amidotransferase activity, Tris/HCl buffer
8
-
-
stable enzyme
8.5
-
-
aminotransferase activity, Tris buffer
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6
10
-
approx. 70% of maximal activity between pH 6 and pH 10
7
9
-
stable enzyme, approx. 65% and unstable enzyme approx. 70% of maximal activity at pH 7, stable enzyme, approx. 75% and unstable enzyme approx. 65% of maximal activity at pH 9
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
-
-
assay at
25
-
-
assay at
37
-
-
assay at
37
-
-
assay at
37
-
-
assay at
38
-
-
assay at
90
-
-
both glutaminase and amidotransferase activity
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
little or no activity in mitochondria or microsomes
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
Bacillus subtilis (strain 168)
Bacillus subtilis (strain 168)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
54000
-
Q9SI61, Q9STG9, Q9T0J5
SDS-PAGE analysis of the recombinant AtGPRAT2 expressed in Escherichia coli
93000
-
-
sucrose density gradient centrifugation, enzyme exists in equilibrium of tetrameric, dimeric and monomeric forms
102000
-
-
sedimentation equilibrium centrifugation, phosphate, phosphoribosyldiphosphate or purine mononucleotides influence the sedimentation profile
110000
-
-
ligand-induced alteration of sedimentation coefficient and Stokes radius, gel filtration or sedimentation equilibrium centrifugation
127000
-
-
liver small form
133000
-
-
placenta small form, large form is converted to small form by incubation with phosphoribosyldiphosphate
170000
-
-
placenta large form, in the presence of AMP or GMP
172000
-
-
sedimentation equilibrium centrifugation in the presence of phosphoribosyldiphosphate, the sedimentation profile is influenced by phosphate, phosphoribosyldiphosphate or purine mononucleotides
180000
-
-
mutant unstable enzyme, gel filtration
181000
-
-
sedimentation equilibrium centrifugation in the presence of phosphate, the sedimentation profile is influenced by phosphate, phosphoribosyldiphosphate or purine mononucleotides
185000
-
-
highly concentrated enzyme solution, sucrose density gradient centrifugation, enzyme exists in equilibrium of tetramer, dimer and monomeric forms, conversion of dimer to tetramer within 10fold increase in protein concentration
194000
-
-
sedimentation equilibrium centrifugation
195000
-
-
sucrose density gradient centrifugation
200000
-
-
highly concentrated enzyme solution, gel filtration, enzyme exists in equilibrium of tetrameric, dimeric and monomeric forms, conversion of dimer to tetramer within 10fold increase in protein concentration, AMP and GMP stabilize the dimeric form, GDP stabilizes the tetrameric form
200000
-
-
native PAGE
200000
-
-
ligand-induced alteration of sedimentation coefficient and Stokes radius in the presence of phosphoribosyldiphosphate or phosphate, gel filtration and sedimentation equilibrium centrifugation
215000
-
-
gel filtration
215000
-
-
gel filtration; only one enzyme form
224000
-
-
gel filtration
270000
-
-
placenta, large form, small form is converted to large form by incubation with purine nucleotides, large form presumably catalytically inactive
292000
-
-
liver, large form
360000
-
-
wild-type stable enzyme, gel filtration
8000000
-
-
probably an aggregation of several purine synthetic activities, gel filtration
additional information
-
-
the smaller molecular weight form of the liver enzyme is observed when incubated with purine nucleotides, the smaller one when incubated with phosphoribosyldiphosphate
additional information
-
-
enzymes from human placenta, Chinese hamster fibroblasts and mouse liver exist in two molecular weight forms, the larger one is observed when incubated with purine nucleotides, the smaller one when incubated with phosphoribosyldiphosphate
additional information
-
-
-
additional information
-
-
the smaller molecular weight form of the liver enzyme is observed when incubated with purine nucleotides, the smaller one when incubated with phosphoribosyldiphosphate
additional information
-
-
partially purified enzyme shows 3 molecular forms: an inactive tetramer formed in the presence of AMP, an active dimer formed with 5-phospho-alpha-D-ribose 1-diphosphate and an inactive dimer formed with piritrexim
additional information
-
-
2 molecular forms: homodimeric in the presence of 5-phospho-alpha-D-ribose 1-diphosphate, homotetrameric in the presence of purine ribonucleotides
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
3-4 * 57000, SDS-PAGE
?
-
2-4 * 50000, SDS-PAGE
?
-
3-4 * 56395, calculated from nucleotide sequence; 3-4 * 57000, SDS-PAGE
?
-
x * 58000, SDS-PAGE
?
-
x * 56700, calculated
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
when mapped onto the structure from Bacillus subtilis GPRAT (PDB: 1AO0), the site of mutation R264K lies within 10 A of the glutaminase site
Q9SI61, Q9STG9, Q9T0J5
crystal structure at 3.0 A
-
crystals of the ternary enzyme-ADP-GMP complex are grown in glass melting point capillaries by the microbath method, 20 mg/ml enzyme are incubated with 1 mM ADP, 1 mM GMP, and 5 mM MgCl2 and mixed with an equal volume of 24% polyethylene glycol 8000, 200 mM KCl, 50 mM N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid, pH 7.9, brown crystals emerge after 6-12 weeks
P00497
crystal structure in the absence of ligands at 2.0 A, and with bound AMP at 2.5 A
-
crystal structure of 6-diazo-5-oxonorleucine inactivated enzyme at 2.3 A resolution
-
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
37
-
-
glutaminase half-life: 25 h
48
-
-
wild-type, stable enzyme from cells of mid-exponential growth phase, t1/2: 34 min, from cells of stationary growth phase, t1/2: 2-5 min, mutant enzyme, t1/2: 2 min, in the presence of ADP or adenosine 3 min, and 4 min in the presence of IMP
60
-
-
6 min, about 25% loss of activity; after 3-6 min loss of ATP-sensitivity
60
-
-
at least 15 min stable
80
-
-
glutaminase half-life: 65 min
additional information
-
-
AMP and GMP enhance thermal stability
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ADP or ADP and GMP, ratio 1/1, stabilize equilibrium between dimeric and tetrameric form
-
AMP or GMP stabilizes dimeric enzyme form
-
AMP stabilizes against inactivation by O2
-
GDP stabilizes tetrameric enzyme form
-
phosphoribosyldiphosphate and other nucleotides antagonize stabilizing effect of AMP
-
2-mercaptoethanol stabilizes during purification
-
prolonged dialysis against distilled water leads to precipitation
-
stable during all stages of purification provided thiols and oxygen are avoided
-
freezing inactivates, not stabilized by 30% glycerol
-
high concentrations of thiol reagents stabilize during purification
-
Mg2+ and phosphate are essential for stability
-
stability profile depends on growth rate and growth phase of cell culture, PMSF improves stability
-
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
anaerobic conditions stabilize
-
489748
in vitro O2 oxidizes iron in the Fe-S cluster to the high spin ferric state and sulfur to a mixtur of S in thiocystine residues plus other unidentified products
-
489755
O2, rather than peroxide, superoxide, hydroxyl radical or singlet oxygen, inactivates, allosteric inhibitors, such as AMP, ADP, GMP or GDP modulate the rate of inactivation
-
489753
oxygen-labile in vivo and in vitro, AMP protects
-
489748
oxygen sensitive enzyme
-
489754
anaerobic conditions stabilize
-
489747
low concentrations of thiols e.g. dithiothreitol or 2-mercaptoethanol accelerate aerobic inactivation
-
489747
oxygen inactivates during purification
-
489747
oxygen sensitive enzyme
-
489754
amido- and aminotransferase activity is lost after exposure to oxygen
-
489754
oxygen sensitive enzyme
-
489754
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
2-mercaptoethanol and phosphoribosyldiphosphate stabilize during storage at 4C
-
2-mercaptoethanol stabilizes during storage
-
4C, in phosphate buffer, pH 7.4, 60 mM 2-mercaptoethanol and phosphoribosyldiphosphate, at least 10 days
-
phosphate stabilizes during storage
-
storage in 25 mM Tris buffer inactivates with 26% or 4% residual activity after 6 or 10 days, respectively
-
4C, 25% loss of activity per day
-
-20C or 4C, cell-free extract, overnight, more than 50% loss of activity
-
-20C, crude, more than 50% loss of activity overnight
-
4C, 50 mM phosphate bufer, pH 7.4, 5 mM MgCl2, 60 mM 2-mercaptoethanol, at least 4 weeks, no loss of activity
-
4C, partially purified preparation in 50 mM phosphate buffer, pH 7.4, 5 mM Mg2+ and 60 mM 2-mercaptoethanol, at least 4 weeks
-
4C, phosphate buffer, 10 d, 40% loss of activity
-
3C, crude mutant enzyme extract, inactivation overnight, partially purified mutant enzyme: inactivation within 72 h, PMSF stabilizes
-
3C, crude or partially purified wild-type enzyme, at least 72 h stable
-
PMSF stabilizes during storage
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant enzyme
-
from bacterial extracts by desalting on a Bio-Rad 10DG column in presence of 10 mM dithiothreitol, stored at -70C
Q9SI61, Q9STG9, Q9T0J5
partial
-
partial, ATP-agarose affinity chromatography
-
protamine sulfate, DEAE-cellulose
-
ammonium sulfate, heat treatment, calcium phosphate gel, DEAE-cellulose; partial
-
ammonium sulfate, heat treatment, DEAE-Sepharose, Blue Dextran-Sepharose, hydroxylapatite
-
recombinant wild-type and P410W mutant enzyme
-
hexa-His-tagged mutant I198V and its variant 1-04 by metal affinity chromatography in presence of 1 mM beta-mercaptoethanol and 10% (v/v) glycerol, elution with 250 mM imidazole followed by dialysis
P0AG16
Sepharose CL-2B, glycerol gradient
-
DEAE-cellulose, partial purification
-
partial
-
heat treatment, acid precipitation, ammonium sulfate, ethanol, Sephadex G-150, DE-52
-
streptomycin, ammonium sulfate, Sephadex g-200
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression in Escherichia coli
-
amplified from seedling cDNA library to encode 11-amino acid autoprocessing propeptide, N-terminal cysteine for an active enzyme and deleted chloroplast transit peptide, in pCR2.1TOPO for sequencing, in pET26b for expression in Escherichia coli BL21(DE3); amplified from seedling cDNA library to encode 11-amino acid autoprocessing propeptide, N-terminal cysteine for an active enzyme and deleted chloroplast transit peptide, in pCR2.1TOPO for sequencing, in pET26b for expression in Escherichia coli BL21(DE3); amplified from seedling cDNA library to encode 11-amino acid autoprocessing propeptide, N-terminal cysteine for an active enzyme and deleted chloroplast transit peptide, in pCR2.1TOPO for sequencing, in pET26b for expression in Escherichia coli BL21(DE3)
Q9SI61, Q9STG9, Q9T0J5
expression in Escherichia coli
-
used in gene replacment and complementation experiments
-, F2XMV3
expression of wild-type, Y74A, Y258A, Y258F, K326Q, Y329A, G331I, N351A and Y465A mutant enzyme in Escherichia coli
-
overexpression of wild-type and P410W mutant enzyme in Escherichia coli
-
wildtype, mutant I198V and variants 1-04 and 2-02 of mutant I198V in pCA24N for expression as N-terminal hexa-His-tagged, C-terminal GFP-tagged proteins in Escherichia coli JMB9 DELTA TrpF (auxotrophic for tryptophan) and/or Escherichia coli KK8(pDM), mutant I198V in pCDF-1b for error-prone PCR based mutagenesis yielding variant 1-04
P0AG16
expression of wild-type and K338Q/P422W mutant enzyme in amidophosphoribosyltransferase deficient CHO ade -A cells and in transgenic mice
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
R264K
Q9SI61, Q9STG9, Q9T0J5
increased resistance to DAS734 with >500-fold higher IC50 compared to wild-type: no inhibition by 100 microM DAS734 and no effect on reaction time course, the same allosteric inhibition by adenine nucleotides as the wild-type, Arg-264 conserved in almost every GPRAT sequence
A417W
-
moderate resistance to inhibition by ATP or GPT
D310V
-
almost resistant to inhibition by ATP or GTP, low concentrations of ATP plus GTP activate
G331I
-
50% of wild-type glutaminase activity, reduced inhibition by GMP, enhanced inhibition by AMP
K326Q
-
similar glutaminase and amidotransferase activity as wild-type, not inhibited by GMP
K326Q
-
insensitive to inhibition by GMP, reduced synergistic inhibition
K326Q/P410W
-
binding of GMP and AMP is abolished resulting in loss of inhibition
L415A
P0AG16
mutant has reduced transfer efficiency. The L415A GPATase mutant-thioester-5'-phosphoribosylpyrophosphate complex after 39 ps, shows the leakage of ammonia into bulk solution
N351A
-
approx. 50% of wild-type glutaminase activity, completely insensitive to inhibition by GMP, partially resistent to inhibition by AMP
P410W
-
reduced inhibition by AMP, strong synergistic inhibition by AMP and GMP
R26H
-
extremely labile enzyme
Y258A
-
complete loss of glutaminase and amidotransferase activity
Y258F
-
approx. 50% loss of glutaminase activity, very weak amidotransferase activity
Y329A
-
normal glutaminase activity, approx. 20% amidotransferase activity, less sensitive to GMP inhibition than wild-type
Y465A
-
100% of wild-type glutaminase activity, 28% of wild-type amidotransferase activity, inhibition by GMP and AMP is similar to wild-type
Y74A
-
complete loss of glutaminase activity, little loss of amidotransferase activity
I198V
P0AG16
by missense mutation A592G, purF(I198V) and variant purF(1-04) in pCA24N by two polymerase error prone PCR strategy (1. mutazyme, 2. Taq DNA polymerase in Thermopol reaction buffer) followed by second directed mutagenesis on purF(1-04) by passaging through Escherichia coli mutator strain XL1-Red leading to variant purF(2-02), mutant I198V and variants 1-04 and 2-02 improved growth of Escherichia coli JMB9 DELTA TrpF (auxotrophic for tryptophan) on medium lacking tryptophan by gained phosphoribosylanthranilate isomerase activity
N328S
P0AG16
recurring mutation after error-prone PCR, found in variant 1-04 of mutant I198V which also harbours mutations: I37M, A39T, E85G, P88S, N124S, I198V, K282R and one silent, Asn328 is implicated in PRPP binding and a critical residue for improving phosphoribosylanthranilate isomerase activity. Variant 2-02 has the same open reading frame as 1-04 but optimized transcription.
K333Q
-
almost resistant to inhibition by ATP or GTP, low concentrations of ATP plus GTP activate
additional information
-
enzyme deletion mutant, auxotrophic for adenine, produces lower levels of riboflavin than wild-type
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
P0AG16
marginal secondary enzyme activities can dramatically improve the fitness of contemporary organisms