Information on EC 3.6.3.5 - Zn2+-exporting ATPase

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

EC NUMBER
COMMENTARY hide
3.6.3.5
-
RECOMMENDED NAME
GeneOntology No.
Zn2+-exporting ATPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + H2O + Zn2+/in = ADP + phosphate + Zn2+/out
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
transmembrane transport
SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (Zn2+-exporting)
A P-type ATPase that undergoes covalent phosphorylation during the transport cycle. This enzyme also exports Cd2+ and Pb2+.
CAS REGISTRY NUMBER
COMMENTARY hide
9000-83-3
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
UniProt
Manually annotated by BRENDA team
Salmonella enterica serovar typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + H2O + Ag+/in
ADP + phosphate + Ag+/out
show the reaction diagram
-
-
-
?
ATP + H2O + Ca2+/out
ADP + phosphate + Ca2+/in
show the reaction diagram
-
-
-
-
?
ATP + H2O + Cd2+/in
ADP + phosphate + Cd2+/out
show the reaction diagram
ATP + H2O + Cd2+/out
ADP + phosphate + Cd2+/in
show the reaction diagram
ATP + H2O + Co2+/in
ADP + phosphate + Co2+/out
show the reaction diagram
ATP + H2O + Cu2+/in
ADP + phosphate + Cu2+/out
show the reaction diagram
ATP + H2O + Cu2+/out
ADP + phosphate + Cu2+/in
show the reaction diagram
ATP + H2O + Fe3+/out
ADP + phosphate + Fe3+/in
show the reaction diagram
-
-
-
-
?
ATP + H2O + Hg2+/in
ADP + phosphate + Hg2+/out
show the reaction diagram
-
-
-
?
ATP + H2O + Mn2+/out
ADP + phosphate + Mn2+/in
show the reaction diagram
-
-
-
-
?
ATP + H2O + Ni2+/in
ADP + phosphate + Ni2+/out
show the reaction diagram
ATP + H2O + Pb2+/in
ADP + phosphate + Pb2+/out
show the reaction diagram
ATP + H2O + Zn2+/in
ADP + phosphate + Zn2+/out
show the reaction diagram
ATP + H2O + Zn2+/out
ADP + phosphate + Zn2+/in
show the reaction diagram
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + H2O + Cd2+/in
ADP + phosphate + Cd2+/out
show the reaction diagram
ATP + H2O + Cd2+/out
ADP + phosphate + Cd2+/in
show the reaction diagram
-
-
-
-
?
ATP + H2O + Co2+/in
ADP + phosphate + Co2+/out
show the reaction diagram
-
-
-
-
?
ATP + H2O + Cu2+/in
ADP + phosphate + Cu2+/out
show the reaction diagram
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-
-
-
?
ATP + H2O + Cu2+/out
ADP + phosphate + Cu2+/in
show the reaction diagram
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-
-
-
?
ATP + H2O + Fe3+/out
ADP + phosphate + Fe3+/in
show the reaction diagram
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-
-
-
?
ATP + H2O + Mn2+/out
ADP + phosphate + Mn2+/in
show the reaction diagram
-
-
-
-
?
ATP + H2O + Ni2+/in
ADP + phosphate + Ni2+/out
show the reaction diagram
-
-
-
-
?
ATP + H2O + Pb2+/in
ADP + phosphate + Pb2+/out
show the reaction diagram
ATP + H2O + Zn2+/in
ADP + phosphate + Zn2+/out
show the reaction diagram
ATP + H2O + Zn2+/out
ADP + phosphate + Zn2+/in
show the reaction diagram
additional information
?
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The enzyme mediates active extrusion of Zn2+, which occurred during the exponential phase of growth. The effluxed Zn2+ are not ejected out of the cell but stored in the outer membrane and periplasm.
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ag+
-
activates ATPase activity
Cs2+
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ZntA confers resistance specifically to Pb2+, Zn2+, and Cd2+ in Escherichia coli. Wild-type ZntA binds two metal ions with high affinity, one in the N-terminal domain and another in the transmembrane domain
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thapsigargin
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-
vanadate
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,2-dimyristoyl-phosphatidylglycerol
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solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. Among the most abundant naturally accuring phospholipids, only phosphatidyl-glycerol enhances the in vitro ATPase activity of TntA. Relipidation of detergent-purified ZntA with 1,2-dioleoylphosphatidyl-glycerol increases the ATPase activity 4fold compared to the purified state. Among the phosphatidylglycerol family, highest activity is observed for 1,2-dioleoyl-phosphatidylglycerol followed by 1,2-dimyristoyl-phosphatidylglycerol, 1,2-dipalmitoyl-phosphatidylglycerol and 1,2-distearoyl-phosphatidylglycerol
1,2-dioleoyl-phosphatidylglycerol
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solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. Among the most abundant naturally accuring phospholipids, only phosphatidyl-glycerol enhances the in vitro ATPase activity of TntA. Relipidation of detergent-purified ZntA with 1,2-dioleoylphosphatidyl-glycerol increases the ATPase activity 4fold compared to the purified state. Among the phosphatidylglycerol family, highest activity is observed for 1,2-dioleoyl-phosphatidylglycerol followed by 1,2-dimyristoyl-phosphatidylglycerol, 1,2-dipalmitoyl-phosphatidylglycerol and 1,2-distearoyl-phosphatidylglycerol
1,2-dipalmitoyl-phosphatidylglycerol
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solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. Among the most abundant naturally accuring phospholipids, only phosphatidyl-glycerol enhances the in vitro ATPase activity of TntA. Relipidation of detergent-purified ZntA with 1,2-dioleoylphosphatidyl-glycerol increases the ATPase activity 4fold compared to the purified state. Among the phosphatidylglycerol family, highest activity is observed for 1,2-dioleoyl-phosphatidylglycerol followed by 1,2-dimyristoyl-phosphatidylglycerol, 1,2-dipalmitoyl-phosphatidylglycerol and 1,2-distearoyl-phosphatidylglycerol
1,2-distearoyl-phosphatidylglycerol
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solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. Among the most abundant naturally accuring phospholipids, only phosphatidyl-glycerol enhances the in vitro ATPase activity of TntA. Relipidation of detergent-purified ZntA with 1,2-dioleoylphosphatidyl-glycerol increases the ATPase activity 4fold compared to the purified state. Among the phosphatidylglycerol family, highest activity is observed for 1,2-dioleoyl-phosphatidylglycerol followed by 1,2-dimyristoyl-phosphatidylglycerol, 1,2-dipalmitoyl-phosphatidylglycerol and 1,2-distearoyl-phosphatidylglycerol
L-cysteine
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activates the enzyme and mutant lacking 100 residues
Phospholipid
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solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. Among the most abundant naturally accuring phospholipids, only phosphatidyl-glycerol enhances the in vitro ATPase activity of TntA. Relipidation of detergent-purified ZntA with 1,2-dioleoylphosphatidyl-glycerol increases the ATPase activity 4fold compared to the purified state. Among the phosphatidylglycerol family, highest activity is observed for 1,2-dioleoyl-phosphatidylglycerol followed by 1,2-dimyristoyl-phosphatidylglycerol, 1,2-dipalmitoyl-phosphatidylglycerol and 1,2-distearoyl-phosphatidylglycerol
additional information
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stimulated by an outwardly directed proton gradient
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0026 - 0.075
ATP
0.115
Cd(thiolate)2
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-
-
0.0031 - 0.152
Cd2+
0.004 - 0.133
Co2+
0.0034 - 0.171
Cu2+
0.0021 - 0.169
Ni2+
0.118
Pb(thiolate)2
-
-
-
0.005 - 0.15
Pb2+
0.109
Zn(thiolate)2
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-
0.0051 - 0.2288
Zn2+
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
300
ATP
Noccaea caerulescens
Q70LF4
in the presence of Zn2+ or Cd2+, in 0.6 M Tris, 2 M NaCl and 0.1 M MgCl2, pH 8.0, at 30C
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0681
Cd2+
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-
0.0327
Cu2+
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-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.001242
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-
0.001584
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pH condition equal, pH = 7.0/7.0
0.001668
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pH condition equal, pH = 5.0/5.0
0.001866
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pH condition outward gradient, pH = 5.0/7.0
0.001916
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carrier transport is stimulated by the addition of 1 mM ATP
0.002082
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pH condition inward gradient, pH = 7.0/5.0
additional information
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The activity with Ni2+, Co2+ and Cu2+ are extremely low, 10-20fold lower compared with the values obtained with Pb2+, Zn2+ and Cd2+.
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
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Cd(II)-dependent activity
6 - 6.5
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Pb(II)-dependent and Zn(II)-dependent activity
6
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ATPase activity assay
7.2
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Zn2+ transport measurement at room temperature
7.5
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ATPase activity assay
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
23
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Zn2+ transport measurement at room temperature
37
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ATPase activity assay
additional information
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phosphorylation assays are carried out on ice and room temperature
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
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expression pattern of HMA1 invarious tissues of wild-type plants, HMA1 is expressed preferentially in shoots, including rosette leaves, cauline leaves, ?owers and stems, but little expression is observed in the roots, overview
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Shigella sonnei (strain Ss046)
Shigella sonnei (strain Ss046)
Synechocystis sp. (strain PCC 6803 / Kazusa)
Synechocystis sp. (strain PCC 6803 / Kazusa)
Synechocystis sp. (strain PCC 6803 / Kazusa)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36000
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lipoprotein, SDS-PAGE
50000
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second subunit that contains the phosphorylation site, determined by SDS-PAGE and Western-blotting
69000
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the mutant lacking the N-terminal domain and both the CCCDXXC and GXXCXXC, SDS-PAGE
92000
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1 * 92000, also detection of a dimeric form of 190000 Da, SDS-PAGE; 2 * 92000, also detection of a monomeric form of 92000 Da, SDS-PAGE
128000
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predicted protein size
160000
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dimeric form, SDS-PAGE
additional information
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on PAA gels the protein is present in three forms, as a monomer with an apparent mass of 92 kDa, as a dimer of 190 kDa and as a proteolytic fragment of 67 kDa
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 60000, SDS-PAGE
monomer
additional information
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 20% glycerol, 2 mM beta-mercaptoethanol, 0.5 mM PMSF
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-80C, 100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM Tris(2-carboxyethyl)phosphine hydrochoride
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
isolated from roots, purified via metal affinity chromatography
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lipoprotein MtsA
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lysosomal membrane vesicles are prepared
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membranes from yeast are prepared, purification of the HMA2 C-terminal metal binding domain is performed using a Strep-Tactin Superflow column
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mutant enzymes C59A/C62A and C392A/C394A
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Ni-IDA metal affinity column chromatography, gel filtration
Ni2+-nitrilotriacetic acid resin column chromatography
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purification of the HMA2 N-terminal metal binding domain is performed using Strep-tag affinity chromatography, membranes from HMA2 expressing yeasts are prepared
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Talon resin column chromatography
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the membrane fractions of the cells are isolated
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the recombinant mutant lacking the N-terminal domain and both the CCCDXXC and GXXCXXC
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using a Talon-affinity column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Arabidopsis thaliana HMA2 and truncated forms of the protein are prepared using the bacterial expression vector pPRIBA1 and the yeast expression vector pYES2/CT
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expressed in Escherichia coli C43 (DE3) cells
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expressed in Escherichia coli Top10 cells
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expression in Escherichia coli
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expression in Saccharomyces cerevisiae
expression of wild-type HMA2 and mutant derivatives in transgenic hma2,hma4, Zn-deficient plants from the HMA2 promoter, quantitative realtime PCR expression analysis
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HMA1 DNA and amino acid sequence determination and analysis, phylogenetic analysis, quantitative realtime RT-PCR expression analysis, expression in Saccharomyces cerevisiae
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identification of two independent mutant alleles for both genes, HMA2 and HMA4. The hma2-2 mutant is indistinguishable from the wild-type, whereas the hma4-1 and the double mutant hma2-2,hma4-1 accumulate approximately 2fold and 4fold less Zn, respectively, than the wild type.
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into the pBAD/Myc-Hic C or pBAD/Myc-His A vector for expression in the Escherichia coli strain LMG194(zntA::cat)
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into the pTRCHisA vector for expression in the Escherichia coli TOP 10 strain
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mutant enzymes C59A/C62A and C392A/C394A
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The cadA and zntA genes are located on the bacterial chromosome. Expression of zntA in Escherichia coli predominantly mediated resistance to zinc, and expression of cadA predominantly mediated resistance to cadmium.
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the PCR-amplified cadA gene from Geobacillus stearothermophilus is cloned into the expression vector pBAD-TOPO
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the yeast expression vector pYES2/CT carrying Arabidopsis thaliana HMA2 containing a C-terminal Strep-tag is prepared, the HMA2 N-terminal metal binding domain is cloned into the pPRIBA1 vector for expression in Escherichia coli BL21DE3pLysS cells
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
after zinc shock (0.05 mM Zn2+), the mRNA level increases substantially in a zinc-dependent manner
expression of the zntA gene is inducible by CdCl2, ZnCl2 and CoCl2, of which CdCl2 is the most potent inducer. The metal-induced expression of zntA is controlled by the MerR-like regulator ZntR
there is no significant difference in HMA2 expression after Cd2+ or Zn2+ treatment
treatment with 18 microM of zinc for 1 h, increases NpunR4017 mRNA levels by up to 1300% above basal levels
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C-MBD
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HMA2 C-terminal metal binding domain
C17A
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mutant, 36% decrease in Vmax
C18A
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mutant, 34% decrease in Vmax
C357G
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mutant enzyme does not confer Cd and Zn resistance to yeast
DELTAC-HMA2
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mutant, lacking the 244 C-terminal amino acids
DELTAN-HMA2
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mutant, lacking the N-terminal first 75 amino acids, 56% decrease in Vmax
DELTANC-HMA2
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mutant, lacking the N-terminal first 75 amino acids and the 244 C-terminal amino acids
E21A
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mutant, 43% decrease in Vmax
E21C
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mutant, 60% decrease in Vmax
S20A
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mutant, shows Vmax similar to that of wild-type HMA2
S20C
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mutant, shows Vmax similar to that of wild-type HMA2
A508F
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at low ATP concentrations the mutant enzyme is poorly phosphorylated
C392A
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mutant for evaluating the importance of the cysteine residue of the conserved 392CPC394 motif in metal ion binding
C392A/C394A
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mutant enzyme binds metal ions only at the N-terminal site
C392H
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mutant for evaluating the importance of the cysteine residue of the conserved 392CPC394 motif in metal ion binding
C392H/C394H
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mutant for evaluating the importance of the cysteine residue of the conserved 392CPC394 motif in metal ion binding
C392S
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mutant for evaluating the importance of the cysteine residue of the conserved 392CPC394 motif in metal ion binding
C392S/C394S
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mutant for evaluating the importance of the cysteine residue of the conserved 392CPC394 motif in metal ion binding
C394A
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mutant for evaluating the importance of the cysteine residue of the conserved 392CPC394 motif in metal ion binding
C394S
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mutant for evaluating the importance of the cysteine residue of the conserved 392CPC394 motif in metal ion binding
C59A/C62A
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mutant enzyme in which the N-terminal metal-binding site is disabled by site-specific mutagenesis, can bind only one metal ion
D436N
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the mutant is completely inactive with respect to both in vivo resistance and ATP hydrolysis activity
D714A
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mutant enzyme is not able to confer resistance to Pb2+, Zn2+, and Cd2+ salts, large reduction in ATPase activity
D714E
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mutant enzyme is not able to confer resistance to Pb2+, Zn2+, and Cd2+ salts, large reduction in ATPase activity, retains the ability to bind metal ions with high affinity at the transmembrane site
D714H
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mutant enzyme is not able to confer resistance to Pb2+, Zn2+, and Cd2+ salts, large reduction in ATPase activity, retains the ability to bind metal ions with high affinity at the transmembrane site
D714M
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significant metal-independent ATPase activity, poor phosphorylation by ATP and Pi
D714P
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mutant enzyme is not able to confer resistance to Pb2+, Zn2+, and Cd2+ salts, large reduction in ATPase activity
E470A
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metal ion-stimulated activity is reduced, about 30-40%. Mutant is phosphorylated more strongly in comparison to the wild type enzyme
G444V
-
mutation stabilizes the E2-P state. Gly144 might become close to P upon domain movements
G503S
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at low ATP concentrations the mutant enzyme is poorly phosphorylated. The phosphorylation defect of the mutant enzyme can be partially compensated by using higher ATP concentrations
G505R
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at low ATP concentrations the mutant enzymeis poorly phosphorylated. The phosphorylation defect of the mutant enzyme can be fully compensated by using higher ATP concentrations
H475A
-
mutant enzyme reacts poorly with ATP, mutation influences the binding of ATP and also other catalytic steps
H475D
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mutant enzyme reacts poorly with ATP, mutation influences the binding of ATP and also other catalytic steps
H475L
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mutant enzyme reacts poorly with ATP, mutation influences the binding of ATP and also other catalytic steps
H475Q
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metal ion-stimulated activity is reduced, about 30-40%. Mutant is phosphorylated more weakly in comparison to the wild type enzyme. Mutation affects the reaction with ATP and phosphate and stabilizes the enzyme in a dephosphorylated state
H475S
-
mutant enzyme reacts poorly with ATP, mutation influences the binding of ATP and also other catalytic steps
K693N
-
very low activity, no Cu2+-dependent phosphorylation with ATP, hyperphosphorylation by Pi, altered metal sensitivity of phosphorylation by Pi
K693N/D714M
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very low activity and phosphorylation with ATP, almost normal phosphorylation with Pi, altered metal senstivity of Pi phosphorylation
P393A
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mutant, shows no activity and can not bind any metal ion at the transmembrane site
SAAS
-
mutation of two the two cysteines to serines in the CAAC motif near the N-terminus, ATPase activity 50%, diminished phosphorylation, faster dephosphorylation
SPS
-
mutation of two the two cysteines to serines in the CPC motif in transmembrane helix 6, inactive, very low phosphorylation
additional information
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