BRENDA - Enzyme Database

Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2

Vestergaard, A.L.; Coleman, J.A.; Lemmin, T.; Mikkelsen, S.A.; Molday, L.L.; Vilsen, B.; Molday, R.S.; Dal Peraro, M.; Andersen, J.P.; Proc. Natl. Acad. Sci. USA 111, E1334-E1343 (2014)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
7.6.2.1
phosphatidylserine
stimulates the ATPase activity about 20fold
Bos taurus
Engineering
EC Number
Amino acid exchange
Commentary
Organism
7.6.2.1
E371Q
affinity to phosphatidylserine similar to wild-type
Bos taurus
7.6.2.1
F354A
affinity to phosphatidylserine similar to wild-type
Bos taurus
7.6.2.1
F88A
tendency for inhibition at high substrate concentration
Bos taurus
7.6.2.1
I115A
biphasic phosphatidylserine concentration dependence with inhibition at the highest concentration
Bos taurus
7.6.2.1
I362A
increase in the apparent affinity for phosphatidylserine
Bos taurus
7.6.2.1
I364A
80% of wild-type activity
Bos taurus
7.6.2.1
I364E
3-5fold reduction in affinity for phosphatidylserine
Bos taurus
7.6.2.1
I364F
3-5fold reduction in affinity for phosphatidylserine
Bos taurus
7.6.2.1
I364M
3-5fold reduction in affinity for phosphatidylserine
Bos taurus
7.6.2.1
I364Q
activation phase is followed by an inhibition phase at high phosphatidylserine concentration
Bos taurus
7.6.2.1
I364S
30% of wild-type activity
Bos taurus
7.6.2.1
K374A
affinity to phosphatidylserine similar to wild-type
Bos taurus
7.6.2.1
L112A
mutation does not appreciably affect Vmax, the apparent affinities for the substrates, or the phosphorylation rate
Bos taurus
7.6.2.1
L361A
affinity to phosphatidylserine similar to wild-type
Bos taurus
7.6.2.1
L367A
significant reduction in affinity to phosphatidylserine
Bos taurus
7.6.2.1
N359A
dramatic reduction of Vmax to 9-11% of wild-type
Bos taurus
7.6.2.1
N360A
increase in the apparent affinity for phosphatidylserine
Bos taurus
7.6.2.1
P363A
complete loss of activity
Bos taurus
7.6.2.1
S365A
significant reduction in affinity to phosphatidylserine
Bos taurus
7.6.2.1
T369A
increase in the apparent affinity for phosphatidylserine
Bos taurus
7.6.2.1
V906A
pronounced inhibition at high phosphatidylethanol concentration with only a slight inhibition at high phosphatidylserine concentration
Bos taurus
7.6.2.1
Y358A
affinity to phosphatidylserine similar to wild-type
Bos taurus
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
7.6.2.1
0.016
-
ATP
mutant I364A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C; mutant I364S, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.019
-
ATP
mutant I364Q, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.02
-
ATP
mutant T369A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C; mutant V906A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.023
-
ATP
mutant N360A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.024
-
ATP
mutant I362A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.027
-
ATP
mutant F88A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.036
-
ATP
mutant Y358A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.038
-
ATP
wild-type, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.042
-
ATP
mutant F354A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.045
-
ATP
mutant L112A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.049
-
ATP
mutant E371Q, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C; mutant K374A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.058
-
ATP
mutant L361A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.068
-
ATP
mutant L367A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.073
-
ATP
mutant I115A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.103
-
ATP
mutant V906A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.104
-
ATP
mutant S365A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.105
-
ATP
mutant I364S, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.111
-
ATP
mutant I364E, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.131
-
ATP
mutant I364A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.148
-
ATP
mutant I364M, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.149
-
ATP
mutant I362A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.178
-
ATP
mutant I364F, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.22
-
ATP
mutant N359A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.25
-
ATP
mutant F88A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.288
-
ATP
mutant I364Q, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.292
-
ATP
mutant I364E, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.369
-
ATP
mutant I364M, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.371
-
ATP
wild-type, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.376
-
ATP
mutant I115A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.379
-
ATP
mutant L367A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.394
-
ATP
mutant L112A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.862
-
ATP
mutant S365A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
1.12
-
ATP
mutant I364F, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
2.215
-
ATP
mutant N359A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
7.6.2.1
Bos taurus
C7EXK4
-
-
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
7.6.2.1
ATP + H2O
-
735189
Bos taurus
ADP + phosphate
-
-
-
?
7.6.2.1
ATP + H2O + phosphatidylethanolamine/in
-
735189
Bos taurus
ADP + phosphate + phosphatidylethanolamine/out
-
-
-
?
7.6.2.1
ATP + H2O + phosphatidylserine/in
-
735189
Bos taurus
ADP + phosphate + phosphatidylserine/out
-
-
-
?
7.6.2.1
additional information
during the transport across the membrane, the phosphatidylserine head group passes near residue I364. I364 is critical to the release of the transported lipid into the cytosolic leaflet. Transmembrane segment M4 residue N359 is involved in recognition of the lipid substrate on the exoplasmic side. Residue I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase
735189
Bos taurus
?
-
-
-
-
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
7.6.2.1
phosphatidylserine
stimulates the ATPase activity about 20fold
Bos taurus
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
7.6.2.1
E371Q
affinity to phosphatidylserine similar to wild-type
Bos taurus
7.6.2.1
F354A
affinity to phosphatidylserine similar to wild-type
Bos taurus
7.6.2.1
F88A
tendency for inhibition at high substrate concentration
Bos taurus
7.6.2.1
I115A
biphasic phosphatidylserine concentration dependence with inhibition at the highest concentration
Bos taurus
7.6.2.1
I362A
increase in the apparent affinity for phosphatidylserine
Bos taurus
7.6.2.1
I364A
80% of wild-type activity
Bos taurus
7.6.2.1
I364E
3-5fold reduction in affinity for phosphatidylserine
Bos taurus
7.6.2.1
I364F
3-5fold reduction in affinity for phosphatidylserine
Bos taurus
7.6.2.1
I364M
3-5fold reduction in affinity for phosphatidylserine
Bos taurus
7.6.2.1
I364Q
activation phase is followed by an inhibition phase at high phosphatidylserine concentration
Bos taurus
7.6.2.1
I364S
30% of wild-type activity
Bos taurus
7.6.2.1
K374A
affinity to phosphatidylserine similar to wild-type
Bos taurus
7.6.2.1
L112A
mutation does not appreciably affect Vmax, the apparent affinities for the substrates, or the phosphorylation rate
Bos taurus
7.6.2.1
L361A
affinity to phosphatidylserine similar to wild-type
Bos taurus
7.6.2.1
L367A
significant reduction in affinity to phosphatidylserine
Bos taurus
7.6.2.1
N359A
dramatic reduction of Vmax to 9-11% of wild-type
Bos taurus
7.6.2.1
N360A
increase in the apparent affinity for phosphatidylserine
Bos taurus
7.6.2.1
P363A
complete loss of activity
Bos taurus
7.6.2.1
S365A
significant reduction in affinity to phosphatidylserine
Bos taurus
7.6.2.1
T369A
increase in the apparent affinity for phosphatidylserine
Bos taurus
7.6.2.1
V906A
pronounced inhibition at high phosphatidylethanol concentration with only a slight inhibition at high phosphatidylserine concentration
Bos taurus
7.6.2.1
Y358A
affinity to phosphatidylserine similar to wild-type
Bos taurus
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
7.6.2.1
0.016
-
ATP
mutant I364A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C; mutant I364S, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.019
-
ATP
mutant I364Q, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.02
-
ATP
mutant T369A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C; mutant V906A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.023
-
ATP
mutant N360A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.024
-
ATP
mutant I362A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.027
-
ATP
mutant F88A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.036
-
ATP
mutant Y358A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.038
-
ATP
wild-type, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.042
-
ATP
mutant F354A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.045
-
ATP
mutant L112A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.049
-
ATP
mutant E371Q, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C; mutant K374A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.058
-
ATP
mutant L361A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.068
-
ATP
mutant L367A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.073
-
ATP
mutant I115A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.103
-
ATP
mutant V906A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.104
-
ATP
mutant S365A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.105
-
ATP
mutant I364S, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.111
-
ATP
mutant I364E, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.131
-
ATP
mutant I364A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.148
-
ATP
mutant I364M, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.149
-
ATP
mutant I362A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.178
-
ATP
mutant I364F, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.22
-
ATP
mutant N359A, presence of phosphatidylserine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.25
-
ATP
mutant F88A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.288
-
ATP
mutant I364Q, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.292
-
ATP
mutant I364E, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.369
-
ATP
mutant I364M, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.371
-
ATP
wild-type, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.376
-
ATP
mutant I115A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.379
-
ATP
mutant L367A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.394
-
ATP
mutant L112A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
0.862
-
ATP
mutant S365A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
1.12
-
ATP
mutant I364F, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
7.6.2.1
2.215
-
ATP
mutant N359A, presence of phosphatidylethanolamine, K0.5 value, pH 7.5, 37°C
Bos taurus
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
7.6.2.1
ATP + H2O
-
735189
Bos taurus
ADP + phosphate
-
-
-
?
7.6.2.1
ATP + H2O + phosphatidylethanolamine/in
-
735189
Bos taurus
ADP + phosphate + phosphatidylethanolamine/out
-
-
-
?
7.6.2.1
ATP + H2O + phosphatidylserine/in
-
735189
Bos taurus
ADP + phosphate + phosphatidylserine/out
-
-
-
?
7.6.2.1
additional information
during the transport across the membrane, the phosphatidylserine head group passes near residue I364. I364 is critical to the release of the transported lipid into the cytosolic leaflet. Transmembrane segment M4 residue N359 is involved in recognition of the lipid substrate on the exoplasmic side. Residue I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase
735189
Bos taurus
?
-
-
-
-