BRENDA - Enzyme Database show

Role of conserved aspartates in the ArsA ATPase

Bhattacharjee, H.; Choudhury, R.; Rosen, B.P.; Biochemistry 47, 7218-7227 (2008)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
7.3.2.7
antimonite
activates 10fold the ATPase activity of wild-type ArsA, activation rates of mutants, overview
Escherichia coli
7.3.2.7
arsenite
activates 3fold the ATPase activity of wild-type ArsA, activation rates of mutants, overview
Escherichia coli
Cloned(Commentary)
EC Number
Commentary
Organism
7.3.2.7
expression of His6-tagged wild-type and mutant ArsA in Escherichia coli strain JM109. At high expression level wild-type ArsA is located in the cytosol, mutants D142A, D142E, and D142N are also found predominantly in the cytosol at similar levels as the wild type, but D447A and D447E proteins are found as insoluble aggregates, while only trace amounts of D447N can be observed in the soluble fraction
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
7.3.2.7
D142A
site-directed mutagenesis, the mutant is activated by arsenite and antimonite in a similar amount as the wild-type enzyme
Escherichia coli
7.3.2.7
D142E
site-directed mutagenesis, the mutant is stronger activated by arsenite and antimonite compared to the wild-type enzyme
Escherichia coli
7.3.2.7
D142N
site-directed mutagenesis, the mutant is activated by arsenite and antimonite in a similar amount as the wild-type enzyme
Escherichia coli
7.3.2.7
D447A
site-directed mutagenesis, the mutant is less activated by arsenite and antimonite compared to the wild-type enzyme
Escherichia coli
7.3.2.7
D447E
site-directed mutagenesis, the mutant is less activated by arsenite and antimonite compared to the wild-type enzyme
Escherichia coli
7.3.2.7
D447N
site-directed mutagenesis, the near complete insolubility of D447N ArsA precludes its purification and biochemical characterization
Escherichia coli
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
7.3.2.7
Trypsin
trypsin cleaves the ArsA at Arg290 to produce a 32 kDa A1 fragment that is catalytically inactive and remains stable to trypsin digestion, and a slightly smaller A2 fragment which is digested rapidly. The trypsin digestion pattern is much different when all three ligands, ATP, Sb(III), and Mg2+, are added together, conditions that produce activated catalysis, overview
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
7.3.2.7
additional information
-
additional information
activation kinetics of wild-type and mutant enzymes, overview
Escherichia coli
7.3.2.7
0.035
-
ATP
recombinant wild-type enzyme
Escherichia coli
7.3.2.7
0.15
-
ATP
recombinant mutant D142E
Escherichia coli
7.3.2.7
0.2
-
ATP
recombinant mutant D447E
Escherichia coli
7.3.2.7
0.6
-
ATP
recombinant mutant D447A
Escherichia coli
7.3.2.7
0.9
-
ATP
recombinant mutant D142N
Escherichia coli
7.3.2.7
1.25
-
ATP
recombinant mutant D142A
Escherichia coli
Localization
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
7.3.2.7
membrane
-
Escherichia coli
16020
-
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
7.3.2.7
Mg2+
required, Asp142 is involved in Mg2+ binding
Escherichia coli
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
7.3.2.7
ATP + H2O + arsenite/in
Escherichia coli
-
ADP + phosphate + arsenite/out
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
7.3.2.7
Escherichia coli
-
gene arsA
-
Purification (Commentary)
EC Number
Commentary
Organism
7.3.2.7
recombinant His6-tagged wild-type and mutant ArsA from Escherichia coli strain JM109 to over 95% homogeneity by nickel affinity chromatography
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
7.3.2.7
ATP + H2O + arsenite/in
-
696278
Escherichia coli
ADP + phosphate + arsenite/out
-
-
-
?
7.3.2.7
ATP + H2O + arsenite/in
Asp142 is involved in Mg2+ binding and also plays a role in signal transduction between the catalytic and activation domains. In contrast, Asp447 is not nearly as critical for Mg2+ binding as Asp142 but appears to be in communication between the metal and catalytic sites
696278
Escherichia coli
ADP + phosphate + arsenite/out
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
7.3.2.7
More
ArsA is composed of two homologous halves A1 and A2, each containing a nucleotide binding domain, and a single metalloid binding or activation domain is located at the interface of the two halves of the protein. The metalloid binding domain is connected to the two nucleotide binding domains through two DTAPTGH sequences, one in A1 and the other in A2. The DTAPTGH sequences are proposed to be involved in information communication between the metal and catalytic sites
Escherichia coli
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
7.3.2.7
ATP
-
Escherichia coli
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
7.3.2.7
antimonite
activates 10fold the ATPase activity of wild-type ArsA, activation rates of mutants, overview
Escherichia coli
7.3.2.7
arsenite
activates 3fold the ATPase activity of wild-type ArsA, activation rates of mutants, overview
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
7.3.2.7
expression of His6-tagged wild-type and mutant ArsA in Escherichia coli strain JM109. At high expression level wild-type ArsA is located in the cytosol, mutants D142A, D142E, and D142N are also found predominantly in the cytosol at similar levels as the wild type, but D447A and D447E proteins are found as insoluble aggregates, while only trace amounts of D447N can be observed in the soluble fraction
Escherichia coli
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
7.3.2.7
ATP
-
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
7.3.2.7
D142A
site-directed mutagenesis, the mutant is activated by arsenite and antimonite in a similar amount as the wild-type enzyme
Escherichia coli
7.3.2.7
D142E
site-directed mutagenesis, the mutant is stronger activated by arsenite and antimonite compared to the wild-type enzyme
Escherichia coli
7.3.2.7
D142N
site-directed mutagenesis, the mutant is activated by arsenite and antimonite in a similar amount as the wild-type enzyme
Escherichia coli
7.3.2.7
D447A
site-directed mutagenesis, the mutant is less activated by arsenite and antimonite compared to the wild-type enzyme
Escherichia coli
7.3.2.7
D447E
site-directed mutagenesis, the mutant is less activated by arsenite and antimonite compared to the wild-type enzyme
Escherichia coli
7.3.2.7
D447N
site-directed mutagenesis, the near complete insolubility of D447N ArsA precludes its purification and biochemical characterization
Escherichia coli
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
7.3.2.7
Trypsin
trypsin cleaves the ArsA at Arg290 to produce a 32 kDa A1 fragment that is catalytically inactive and remains stable to trypsin digestion, and a slightly smaller A2 fragment which is digested rapidly. The trypsin digestion pattern is much different when all three ligands, ATP, Sb(III), and Mg2+, are added together, conditions that produce activated catalysis, overview
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
7.3.2.7
additional information
-
additional information
activation kinetics of wild-type and mutant enzymes, overview
Escherichia coli
7.3.2.7
0.035
-
ATP
recombinant wild-type enzyme
Escherichia coli
7.3.2.7
0.15
-
ATP
recombinant mutant D142E
Escherichia coli
7.3.2.7
0.2
-
ATP
recombinant mutant D447E
Escherichia coli
7.3.2.7
0.6
-
ATP
recombinant mutant D447A
Escherichia coli
7.3.2.7
0.9
-
ATP
recombinant mutant D142N
Escherichia coli
7.3.2.7
1.25
-
ATP
recombinant mutant D142A
Escherichia coli
Localization (protein specific)
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
7.3.2.7
membrane
-
Escherichia coli
16020
-
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
7.3.2.7
Mg2+
required, Asp142 is involved in Mg2+ binding
Escherichia coli
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
7.3.2.7
ATP + H2O + arsenite/in
Escherichia coli
-
ADP + phosphate + arsenite/out
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
7.3.2.7
recombinant His6-tagged wild-type and mutant ArsA from Escherichia coli strain JM109 to over 95% homogeneity by nickel affinity chromatography
Escherichia coli
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
7.3.2.7
ATP + H2O + arsenite/in
-
696278
Escherichia coli
ADP + phosphate + arsenite/out
-
-
-
?
7.3.2.7
ATP + H2O + arsenite/in
Asp142 is involved in Mg2+ binding and also plays a role in signal transduction between the catalytic and activation domains. In contrast, Asp447 is not nearly as critical for Mg2+ binding as Asp142 but appears to be in communication between the metal and catalytic sites
696278
Escherichia coli
ADP + phosphate + arsenite/out
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
7.3.2.7
More
ArsA is composed of two homologous halves A1 and A2, each containing a nucleotide binding domain, and a single metalloid binding or activation domain is located at the interface of the two halves of the protein. The metalloid binding domain is connected to the two nucleotide binding domains through two DTAPTGH sequences, one in A1 and the other in A2. The DTAPTGH sequences are proposed to be involved in information communication between the metal and catalytic sites
Escherichia coli