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Literature summary extracted from

  • Benabdelhak, H.; Schmitt, L.; Horn, C.; Jumel, K.; Blight, M.A.; Holland, I.B.
    Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli (2005), Biochem. J., 386, 489-495.
    View publication on PubMedView publication on EuropePMC

Protein Variants

EC Number Protein Variants Comment Organism
7.4.2.5 D630M ATPase activity is 0.6% of wild-type activity Escherichia coli
7.4.2.5 K508M ATPase activity is 1.3% of wild-type activity Escherichia coli
7.4.2.5 P624C insoluble mutant protein Escherichia coli
7.4.2.5 P624L insoluble mutant protein Escherichia coli
7.4.2.5 P624R insoluble mutant protein Escherichia coli
7.4.2.5 P624S insoluble mutant protein Escherichia coli
7.4.2.5 V548A insoluble mutant protein Escherichia coli

Inhibitors

EC Number Inhibitors Comment Organism Structure
7.4.2.5 KCl no ATPase activity above 300 mM Escherichia coli
7.4.2.5 NaCl no ATPase activity above 300 mM Escherichia coli
7.4.2.5 orthovanadate IC50: 0.016 mM, competitive Escherichia coli
7.5.2.2 Ca2+ 3% activity at pH 8.0 and 10 mM KCl Escherichia coli
7.5.2.2 Co2+ 47% activity at pH 8.0 and 10 mM KCl Escherichia coli
7.5.2.2 additional information the activity is reversibly inhibited by high salt concentrations in the physiologically range accompanied by proportional decreased binding of 8-azido-ATP (3-5% residual activity at 200-300 mM salt) Escherichia coli
7.5.2.2 orthovanadate 0.005-0.05 mM, pH 8.0, IC50: 0.016 mM Escherichia coli
7.5.2.2 Zn2+ 1% activity at pH 8.0 and 10 mM KCl Escherichia coli

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
7.4.2.5 0.65
-
 ATP pH 8.0, 25°C, 10 mM NaCl Escherichia coli
7.4.2.5 1.09
-
 ATP pH 8.0, 25°C, 50 mM NaCl Escherichia coli
7.4.2.5 1.5
-
 ATP pH 8.0, 25°C, 100 mM NaCl Escherichia coli
7.4.2.5 2.33
-
 ATP pH 8.0, 25°C, 200 mM NaCl Escherichia coli
7.5.2.2 0.65
-
 ATP KM-value with 50% of the enzyme being substrate-saturated, in the presence of 10 mM NaCl Escherichia coli
7.5.2.2 1.09
-
 ATP KM-value with 50% of the enzyme being substrate-saturated, in the presence of 50 mM NaCl Escherichia coli
7.5.2.2 1.5
-
 ATP KM-value with 50% of the enzyme being substrate-saturated, in the presence of 100 mM NaCl Escherichia coli
7.5.2.2 2.33
-
 ATP KM-value with 50% of the enzyme being substrate-saturated, in the presence of 200 mM NaCl Escherichia coli

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
7.4.2.5 Co2+ divalent cation required, Mg2+ shows 40% of the activity with Mn2+ Escherichia coli
7.4.2.5 Mg2+ divalent cation required, Mg2+ shows 80% of the activity with Mn2+ Escherichia coli
7.4.2.5 Mn2+ divalent cation required, Mn2+ is most effective Escherichia coli
7.5.2.2 Mg2+ 100% activity at pH 8.0 and with 10 mM KCl Escherichia coli
7.5.2.2 Mn2+ 125% activity at pH 8.0 and with 10 mM KCl Escherichia coli

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
7.5.2.2 27500
-
 HlyB NBD in the presence of 100 mM KCl and ATP, ultracentrifugation Escherichia coli
7.5.2.2 27700
-
 HlyB NBD in the presence of 10 mM KCl and in the absence of ATP, ultracentrifugation Escherichia coli
7.5.2.2 28000
-
 His-tagged HlyB ABC domain, gel filtration Escherichia coli
7.5.2.2 30800
-
 HlyB NBD in the presence of 10 mM KCl and ATP, ultracentrifugation Escherichia coli
7.5.2.2 35700
-
 HlyB NBD in the absence of KCl and ATP, ultracentrifugation Escherichia coli

Organism

EC Number Organism UniProt Comment Textmining
7.4.2.5 Escherichia coli
-
-
-
7.5.2.2 Escherichia coli
-
 strain DH5alpha
-
7.5.2.2 Escherichia coli DH5-alpha
-
 strain DH5alpha
-

Purification (Commentary)

EC Number Purification (Comment) Organism
7.5.2.2 HlyB ABC domain, Ni2+-chelating Sepharose 16/20 fast flow column chromatography, His-tagged HlyB ABC domain, immobilized metal-affinity chromatography Escherichia coli

Storage Stability

EC Number Storage Stability Organism
7.4.2.5 -80°C, the ATPase domain is indefinitly stable in phosphate or Tris buffer, pH 8, in the absence of ATP concentrations of up to 25 mg/ml Escherichia coli
7.4.2.5 4°C, the ATPase domain is stable for at least 2 days in phosphate or Tris buffer, pH 8, in the absence of ATP concentrations of up to 25 mg/ml Escherichia coli
7.5.2.2 -80°C, 50 mM phosphate buffer pH 8, 50 mM KCl and 10% glycerol, 15 mg/ml HlyB ABC domain, indefinite storage duration, no loss of activity Escherichia coli
7.5.2.2 -80°C, phosphate or Tris buffer pH 8, in the absence of ATP, up to 25 mg/ml His-tagged HlyB ABC domain, indefinite storage duration, no loss of activity Escherichia coli
7.5.2.2 25°C, 50 mM phosphate buffer pH 8, 50 mM KCl and 10% glycerol, 15 mg/ml HlyB ABC domain, 24h, 20% loss of activity Escherichia coli
7.5.2.2 4°C, 50 mM phosphate buffer pH 8, 50 mM KCl and 10% glycerol, 15 mg/ml HlyB ABC domain, 2 days, no loss of activity Escherichia coli
7.5.2.2 4°C, phosphate or Tris buffer pH 8, in the absence of ATP, up to 25 mg/ml His-tagged HlyB ABC domain, at least 2 days, no loss of activity Escherichia coli

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
7.4.2.5 ATP + H2O + alpha-haemolysin/in
-
 Escherichia coli ADP + phosphate + alpha-haemolysin/out
-
 ?
7.4.2.5 ATP + H2O + alpha-haemolysin/in
-
 Escherichia coli DH5-alpha ADP + phosphate + alpha-haemolysin/out
-
 ?
7.5.2.2 8-azido-ATP + H2O + ?
-
 Escherichia coli 8-azido-ADP + phosphate + ?
-
 ?
7.5.2.2 8-azido-ATP + H2O + ?
-
 Escherichia coli DH5-alpha 8-azido-ADP + phosphate + ?
-
 ?
7.5.2.2 ATP + H2O + ?
-
 Escherichia coli ADP + phosphate + ?
-
 ?
7.5.2.2 ATP + H2O + ?
-
 Escherichia coli DH5-alpha ADP + phosphate + ?
-
 ?

Subunits

EC Number Subunits Comment Organism
7.4.2.5 monomer under most conditions, only the monomer form can be detected, indicating that dimers are very unstable. Monomers can interact at least transiently and are the important species during ATP hydrolysis Escherichia coli
7.5.2.2 dimer 2 * 28000, gel filtration, small amounts of dimers (apparently when stabilized by 8-azido-ATP) are detected even in the presence of salt Escherichia coli
7.5.2.2 monomer 1 * 28000, gel filtration and ultracentrifugation, over a wide range of protein or NaCl or KCl concentrations the enzyme is only detected as monomer Escherichia coli

Synonyms

EC Number Synonyms Comment Organism
7.4.2.5 HlyB
-
 Escherichia coli
7.5.2.2 ABC ATPase domain of HlyB transporter
-
 Escherichia coli
7.5.2.2 ATP-binding cassette ATPase domain of the haemolysin B transporter
-
 Escherichia coli
7.5.2.2 HlyB NBD
-
 Escherichia coli

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
7.4.2.5 0.11
-
 ATP pH 8.0, 25°C, 200 mM NaCl Escherichia coli
7.4.2.5 0.2
-
 ATP pH 8.0, 25°C, 100 mM NaCl Escherichia coli
7.4.2.5 0.31
-
 ATP pH 8.0, 25°C, 50 mM NaCl Escherichia coli
7.4.2.5 0.33
-
 ATP pH 8.0, 25°C, 10 mM NaCl Escherichia coli
7.5.2.2 0.11
-
 ATP KM-value with 50% of the enzyme being substrate-saturated, in the presence of 200 mM NaCl Escherichia coli
7.5.2.2 0.2
-
 ATP KM-value with 50% of the enzyme being substrate-saturated, in the presence of 100 mM NaCl Escherichia coli
7.5.2.2 0.31
-
 ATP KM-value with 50% of the enzyme being substrate-saturated, in the presence of 50 mM NaCl Escherichia coli
7.5.2.2 0.33
-
 ATP KM-value with 50% of the enzyme being substrate-saturated, in the presence of 10 mM NaCl Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4.2.5 8
-
-
 Escherichia coli
7.5.2.2 8
-
 in the presence of 3 mM MgCl2 and 10 mM KCl Escherichia coli

pH Range

EC Number pH Minimum pH Maximum Comment Organism
7.4.2.5 7 8.5 pH 7.0: about 75% of maximal activity, pH 8.5: about 85% of maximal activity Escherichia coli
7.5.2.2 7 8.5
-
 Escherichia coli

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
7.5.2.2 7
-
 75% activity in the presence of 3 mM MgCl2 and 10 mM KCl Escherichia coli
7.5.2.2 7.5
-
 90% activity in the presence of 3 mM MgCl2 and 10 mM KCl Escherichia coli
7.5.2.2 8
-
 100% activity in the presence of 3 mM MgCl2 and 10 mM KCl Escherichia coli
7.5.2.2 8.5
-
 85% activity in the presence of 3 mM MgCl2 and 10 mM KCl Escherichia coli

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
7.4.2.5 0.016
-
 IC50: 0.016 mM, competitive Escherichia coli orthovanadate
7.5.2.2 0.016
-
 0.005-0.05 mM, pH 8.0, IC50: 0.016 mM Escherichia coli orthovanadate