Information on EC 2.7.2.11 - glutamate 5-kinase

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

EC NUMBER
COMMENTARY hide
2.7.2.11
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RECOMMENDED NAME
GeneOntology No.
glutamate 5-kinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + L-glutamate = ADP + L-glutamate 5-phosphate
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Arginine and proline metabolism
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arginine metabolism
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Biosynthesis of antibiotics
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Carbapenem biosynthesis
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L-citrulline biosynthesis
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L-Ndelta-acetylornithine biosynthesis
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L-ornithine biosynthesis II
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L-proline biosynthesis I
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L-proline biosynthesis III
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Metabolic pathways
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proline metabolism
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SYSTEMATIC NAME
IUBMB Comments
ATP:L-glutamate 5-phosphotransferase
Product rapidly cyclizes to 5-oxoproline and phosphate.
CAS REGISTRY NUMBER
COMMENTARY hide
54596-30-4
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 93151-14
SwissProt
Manually annotated by BRENDA team
strain 93151-14
SwissProt
Manually annotated by BRENDA team
strain CM 25
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
strain PAO 1
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Manually annotated by BRENDA team
strain PAO 1
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Manually annotated by BRENDA team
strain INVDput1pro1
SwissProt
Manually annotated by BRENDA team
salt tolerant cultivar T-44 and salt sensitive variant SML-32
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
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Chlamydomonas reinhardtii gamma-glutamte kinase is functional as demonstrated by successful complementation of Escherichia coli auxotroph JW0232 (strain in which the proB gene has been knocked-out)
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + 5-ethyl-L-glutamate
?
show the reaction diagram
ATP + 5-methyl-L-glutamate
?
show the reaction diagram
ATP + cis-cycloglutamate
ADP + cis-cycloglutamyl phosphate
show the reaction diagram
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
ATP + L-glutamine
?
show the reaction diagram
GTP + L-glutamate
GDP + L-glutamate 5-phosphate
show the reaction diagram
additional information
?
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relation of glutamate kinase activity to cadmium and zinc concentration in the above-ground biomass, regulatory role in plant heavy metal stress adaptation
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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
ATP + L-glutamate
ADP + L-glutamate 5-phosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Cd2+
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increased content in soil decreases enzyme activity
K+
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the enzyme is most active at 30C at a relative high K+ + Na+ concentration and a K+/Na+ ratio of 1.8 to 10.2 and at 0C at both a lower K+ + Na+ concentration and a K+/Na+ ratio
Mn2+
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can partially replace Mg2+
Na+
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the enzyme is most active at 30C at a relative high K+ + Na+ concentration and a K+/Na+ ratio of 1.8 to 10.2 and at 0C at both a lower K+ + Na+ concentration and a K+/Na+ ratio
Zn2+
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increased content in soil decreases enzyme activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5-oxo-L-Pro
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12 mM, 10% inhibition
5-oxoproline
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AAK domain has a crater on the beta sheet C-edge that hosts the active centre and binds 5-oxoproline
As2+
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applied into soil strongly decreases activity by 87.7%
cis-4-hydroxy-L-proline
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DL-3,4-Didehydroproline
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9 mM, 50% inhibition
Hg2+
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0.1 mM, complete inhibition
iodoacetamide
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0.125 mM, 60% inhibition. Preincubation with 0.25 mM dithiothreitol for 5 min partially protects
L-azetidine-2-carboxylic acid
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3 mM, 50% inhibition
L-methionine-DL-sulphoximine
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competitive with L-glutamate
L-Orn
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12 mM, 10% inhibition
L-proline
L-Thioproline
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12 mM, 10% inhibition
methyl L-proline
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-
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Mg2+
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above 20 mM
Mn2+
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above 20 mM
NEM
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0.125 mM, complete inhibition. Preincubation with 0.25 mM dithiothreitol for 5 min partially protects
PCMB
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0.125 mM, complete inhibition. Preincubation with 0.25 mM dithiothreitol for 5 min partially protects
trans-4-hydroxy-L-proline
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additional information
no significant differences in growth between mutant yeast cells on SD agar medium with heat shock (50C, 3 to 5 h), ethanol (16 to20%), and sorbitol (2.0 to 2.5 M) stresses, although the proline-accumulating cells are more resistant to these stresses than cells expressing wild-type GK
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
L-Pro
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at low temperatures the inhibition switches over into allosteric activation and the biosynthesis of proline is started
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.4 - 24.6
ATP
12
L-glutamate
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.06
ADP
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pH 7.0, 37C
0.09 - 310
L-Pro
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08
L-Pro
Triticum aestivum
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IC50: 0.08 mM, at room temperature. At low temperatures the inhibition switches over into allosteric activation and the biosynthesis of proline is started
0.011 - 840
L-proline
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.2
mutant N142D/I166V
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 7
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7.6
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activity assay at, 50 mM phosphate buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8.5
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pH 5.5: about 80% of maximal activity, pH 8.5: about 40% of maximal activity
6 - 7.5
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50% of maximal activity at pH 6.0 and at pH 7.5
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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computer predictions suggest that a chloroplast transit peptide exists at the N-terminal region
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Burkholderia thailandensis (strain E264 / ATCC 700388 / DSM 13276 / CIP 106301)
Campylobacter jejuni subsp. jejuni serotype O:2 (strain ATCC 700819 / NCTC 11168)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42000
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SDS-PAGE
47000
gel filtration
84000
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gel filtration
100000
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SDS-PAGE, recombinant protein
236000
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gamma-glutamyl kinase DHPr, gel filtration
254000
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gel filtration
additional information
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two glutamyl kinases of MW 125000 Da and of 38000 Da are detected by gel filtration on Sephadex G-150, a single glutamyl kinase of 250000 Da is detected by Bio-gel A1.5M chromatpgraphy
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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functional unit of the Escherichia coli enzyme is dimeric and contains an intermolecular hydrogen-bond network that interconnects the active-center cavities of the monomers and is important for substrate binding
hexamer
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6 * 40000, gamma-glutamyl kinase DHPr, SDS-PAGE
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging-drop vapour-diffusion method at 21C in the presence of ADP, MgCl2 and L-glutamate using 1.6 M MgSO4, 0.1 M KCl in 0.1 M MES pH 6.5 as crystallization solution. The tetragonal bipyramid-shaped crystals diffract to 2.5 A resolution using synchrotron radiation. The crystals belong to space group P4(1)(3)2(1)2, with unit-cell parameters a = b = 101.1, c = 178.6 A, and contain two monomers in the asymmetric unit, with 58% solvent content
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in about 4-5 months, using the hanging drop vapour diffusion method, complexed with glutamate and sulfate, or with L-glutamate 5-phosphate, sulfate and 5-oxoproline, at 2.9 A and 2.5 A resolution, belongs to the space groups P41212 or P21, respectively. Dimer of dimers architecture, each subunit contains a 257 residue AAK domain, typical of acylphosphate-forming enzymes, with characteristic alpha3beta8alpha4 sandwich topology, each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic beta5beta4 sandwich fold and three alpha helices
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 9
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722207
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70 - 85
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the enzyme retains approximately 100, 90 and 65% of its activity when heated for 45 min at 70, 80 and 85C, respectively
90
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at 90C, tmGK displays around 40% of the activity when heated for 45 min
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-40C, 40% loss of activity within 2 months, then remains stable
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-70C, stable for several months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
(NH4)2SO4 precipitation and a single ion-exchange chromatography step are used
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by Ni2+ column and gel filtration
enzyme form GK 1
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from strain BRL806, designated as gamma-glutamyl kinase w+ and from reductase-overproducing strain BRL1945, designated as gamma-glutamyl kinase DHPr
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to homogeneity
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using Ni-NTA chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
an artificial bifunctional enzyme, gamma-glutamyl kinase/gamma-glutamyl phosphate reductase obtained by fusing the Escherichia coli genes proA and proB improves NaCl tolerance when expressed in Escherichia coli. The proB gene is fused to the 5'-end of the proA gene with a linker encoding five amino acids
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cloned in pET22 and overexpressed in Escherichia coli
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Escherichia coli DH5alpha strain proB cloning into pET-22b to yield pGKE, and overexpression
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expressed in Escherichia coli as a His-tagged fusion protein
expression in Escherichia coli
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expression of PRO1 in Escherichia coli M15(pREP4)
proB cloning into pET-22b to yield pGKE and overexpression in Escherichia coli BL21(DE3)
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proBA fusion gene and mutated proBA fusion gene expressed in Escherichia coli JM83
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D107A
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mutant shows 40fold increased IC50 (L-proline)
D137A
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mutation hampers proline binding and glutamate binding, IC50 (L-proline) 142fold increased compared to wild-type
D148A
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is active, 150fold increased proline requirement
D148N
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is active, 40fold increased proline requirement
D150A
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is not active
D150N
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is active
D170A
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activity is less than 1% of that of wild-type G5K
D170N
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is active
E135A
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mutation of Glu135 and Lys145 only produce relatively small changes in proline activity, IC50 (L-proline) comparable to wild-type
E143A
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mutant shows an 38fold augmented IC0.5 (L-proline) while kinetic parameters of glutamate and ATP are scarcely changed, IC50 (L-proline) 38fold increased compared to wild-type
E143A/K145A
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mutant shows an enhanced affinity for L-glutamate and increased IC50 (L-proline) compared to wild-type
G51A
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is active
I53A
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decreased kinetic parameters, IC50 (L-proline) increased 5fold compared to wild-type
I69E
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mutation produces a very strong (170fold) decrease on proline activity with no other consequence on the kinetic parameters of the enzyme
K10A
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activity is less than 1% of that of wild-type G5K
K145A
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mutant shows an enhanced affinity for L-glutamate and increased IC50 (L-proline) compared to wild-type
K217A
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activity is less than 1% of that of wild-type G5K, decreased proline requirement
K217R
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is active, decreased proline requirement
M214A
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is active
N134D
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mutation hampers proline binding and glutamate binding, IC50 (L-proline) 76fold increased compared to wild-type
N149A
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activity is less than 1% of that of wild-type G5K, 14fold increased proline requirement
Q100A
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mutant shows drastically reduced catalytic rate and reduced affinity for glutamate, IC50 (L-proline) 3fold decreased compared to wild-type
Q80A
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mutant shows drastically reduced catalytic rate and reduced affinity for glutamate, IC50 (L-proline) 3fold decreased compared to wild-type
R118A
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mutant shows increased affinity for glutamate and reduced L-proline affinity (63fold increased IC50)
R25S/E30K/I193A
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mutant behaves as a dimer in gel filtration experiments, kinetically indistinguishable from wild-type, IC50 (L-proline) comparable to wild-type
S50A
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mutant exhibits a greatly reduced catalytic rate but has a small effect on apparent affinities for glutamate or ATP, IC50 (L-proline) 3fold increased compared to wild-type
T169A
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is active, decreased proline requirement
T169S
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is active, decreased proline requirement
E149K
very insensitive to feedback inhibition, shows prominent increase in the proline content, freeze tolerance like the D154N mutant
I150T
very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
N142D/I166V
very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
S146P
shows prominent increase in the proline content
D154N
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is 64fold less sensitive to feedback inhibition than wild-type GK, leading to proline accumulation
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E149K
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very insensitive to feedback inhibition, shows prominent increase in the proline content, freeze tolerance like the D154N mutant
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I150T
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very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
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N142D/I166V
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very insensitive to feedback inhibition, shows prominent increase in the proline content, is more tolerant to freezing stress than cells expressing the D154N mutant
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S146P
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shows prominent increase in the proline content
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A62T
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drastic reduction in specific activity. 911fold increase in Ki-value for L-Pro compared to wild-type enzyme
A62V
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drastic reduction in specific activity. 188fold increase in Ki-value for L-Pro compared to wild-type enzyme
D147G
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reduction in catalytic activity. 2000fold increase in Ki-value for L-Pro compared to wild-type enzyme
D162G
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drastic reduction in specific activity. 611.1fold increase in Ki-value for L-Pro compared to wild-type enzyme
D162N
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drastic reduction in specific activity. 644fold increase in Ki-value for L-Pro compared to wild-type enzyme
E153A
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reduction in catalytic activity. 555.5fold increase in Ki-value for L-Pro compared to wild-type enzyme
E153G
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reduction in catalytic activity. 555.5fold increase in Ki-value for L-Pro compared to wild-type enzyme
E153K
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reduction in catalytic activity. 3444.4fold increase in Ki-value for L-Pro compared to wild-type enzyme
I149F
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222.2fold increase in Ki-value for L-Pro compared to wild-type enzyme; drastic reduction in specific activity. 222.2fold increase in Ki-value for L-Pro compared to wild-type enzyme
I79T
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reduction in catalytic activity. 21.1fold increase in Ki-value for L-Pro compared to wild-type enzyme
L154S
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reduction in catalytic activity. 1000fold increase in Ki-value for L-Pro compared to wild-type enzyme
M94T
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reduction in catalytic activity. 255.6fold increase in Ki-value for L-Pro compared to wild-type enzyme
S159P
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reduction in catalytic activity. 211.1fold increase in Ki-value for L-Pro compared to wild-type enzyme
D192G
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the mutation causes an enhanced feedback-resistant gamma-glutamyl kinase activity and conferrs an analogue-resistant phenotype to an Escherichia coli transformant containing the mutated gene
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
additional information
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