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Literature summary for 3.4.11.7 extracted from

  • Couvineau, P.; de Almeida, H.; Maigret, B.; Llorens-Cortes, C.; Iturrioz, X.
    Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues (2017), PLoS ONE, 12, e0184237 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 3.4.11.7

Application

Application Comment Organism
drug development development of potent and selective APA inhibitors crossing the blood-brain barrier after oral administration for use as centrally-acting antihypertensive agents Mus musculus

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type and mutant enzymes in CHO-K1 cells Mus musculus

Protein Variants

Protein Variants Comment Organism
additional information Ca2+ activation profile of purified recombinant wild-type and mutated His-mAPAs, using an acidic substrate alpha-L-glutamyl-beta-naphthylamide, overview Mus musculus
R878A site-directed mutagenesis, the mutant shows decreased affinity for the acidic substrate, alpha-L-glutamyl-beta-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+ compared to the wild-type enzyme. Analysis of the 3D models of the Arg878 mutated APAs reveals a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis Mus musculus
R878K site-directed mutagenesis, the mutant shows decreased affinity for the acidic substrate, alpha-L-glutamyl-beta-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+ compared to the wild-type enzyme. Analysis of the 3D models of the Arg878 mutated APAs reveals a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis Mus musculus

Inhibitors

Inhibitors Comment Organism Structure
(4R)-4-amino-5-sulfanylpentanoic acid a linear competitive inhibitor Mus musculus
(S)-3-amino-4-mercapto-butyl sulfonic acid i.e. EC33, a selective APA inhibitor, docking analysis in the presence of Ca2+, the ligand interacts with S1 subsite of Arg-878 in murine APA, three-dimensional structure modelling reavealing a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis, overview Mus musculus
2,6-diaminohexane-1-thiol a linear competitive inhibitor Mus musculus
4-amino-4-phosphonobutyric acid a specific and selective APA inhibitor, a linear competitive inhibitor Mus musculus
methionine-thiol a linear competitive inhibitor Mus musculus
additional information ligand docking study and molecular dynamics simulations with wild-type and mutant enzymes, overview Mus musculus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics Mus musculus
0.0391
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme, with 4 mM Ca2+ Mus musculus
0.165
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mus musculus
0.221
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A, with 4 mM Ca2+ Mus musculus
0.269
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K, with 4 mM Ca2+ Mus musculus
1.5
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A Mus musculus
12.5
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K Mus musculus

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane membrane-bound Mus musculus 16020
-

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ localized at the bottom of the S1 subsite, activates the enzyme and plays a key role of Arg878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis. The Ca2+ atom interacts with the acidic side chains of Asp213 and Asp218, the carbonyl group of Glu215 and three water molecules, one of them being engaged in a hydrogen bond with the negatively charged carboxylate side chains of the inhibitors. Ca2+ activation profile of purified recombinant wild-type and mutated His-mAPAs, using an acidic substrate alpha-L-glutamyl-beta-naphthylamide, overview. Enzyme residue Arg878 does not contribute to Ca2+ binding in the S1 subsite Mus musculus
Zn2+ a zinc metalloprotease, Zn2+ atom is coordinated by the two histidine residues, His385 and His389, of the HEXXH motif, a water molecule, Glu408 of the WLNEG motif and either by one of the oxygen atoms of the phosphate of GluPO3H2, three-dimensional structure modelling Mus musculus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
160000
-
-
 Mus musculus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
angiotensin II + H2O Mus musculus
-
 angiotensin III + L-Asp
-
 ?
cholecystokinin-8 + H2O Mus musculus
-
 ?
-
 ?

Organism

Organism UniProt Comment Textmining
Mus musculus P16406
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from CHO-K1 cells by Co2+ affinity chromatography Mus musculus

Source Tissue

Source Tissue Comment Organism Textmining
brain
-
 Mus musculus
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
alpha-L-aspartate-beta-naphthylamide + H2O high activity Mus musculus L-aspartate + beta-naphthylamine
-
 ?
alpha-L-glutamyl-beta-naphthylamide + H2O best substrate Mus musculus L-glutamine + beta-naphthylamine
-
 ?
angiotensin II + H2O
-
 Mus musculus angiotensin III + L-Asp
-
 ?
cholecystokinin-8 + H2O
-
 Mus musculus ?
-
 ?
L-alanine-beta-naphthylamide + H2O low activity Mus musculus L-alanine + beta-naphthylamine
-
 ?
L-lysine-beta-naphthylamide + H2O low activity Mus musculus L-lysine + beta-naphthylamine
-
 ?
additional information enzyme APA cleaves the N-terminal glutamyl or aspartyl residue from peptide substrates Mus musculus ?
-
 ?

Subunits

Subunits Comment Organism
homodimer 2 * 80000 Mus musculus
More aminopeptidase A (APA) is a 160 kDa homodimeric type II Zn2+ membrane-bound aminopeptidase Mus musculus

Synonyms

Synonyms Comment Organism
aminopeptidase A
-
 Mus musculus
APA
-
 Mus musculus
ENPEP
-
 Mus musculus
mAPA
-
 Mus musculus
type II Zn2+ membrane-bound aminopeptidase
-
 Mus musculus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.12
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K Mus musculus
4.95
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A Mus musculus
13.9
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mus musculus
22.4
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A, with 4 mM Ca2+ Mus musculus
81.7
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme, with 4 mM Ca2+ Mus musculus
135
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K, with 4 mM Ca2+ Mus musculus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
 assay at Mus musculus

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.000074
-
 4-amino-4-phosphonobutyric acid pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme, with 4 mM Ca2+ Mus musculus
0.000118
-
 2,6-diaminohexane-1-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A Mus musculus
0.000268
-
 (S)-3-amino-4-mercapto-butyl sulfonic acid pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme, with 4 mM Ca2+ Mus musculus
0.000314
-
 (4R)-4-amino-5-sulfanylpentanoic acid pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme, with 4 mM Ca2+ Mus musculus
0.000317
-
 2,6-diaminohexane-1-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mus musculus
0.000378
-
 2,6-diaminohexane-1-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K Mus musculus
0.000475
-
 methionine-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A Mus musculus
0.000619
-
 4-amino-4-phosphonobutyric acid pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K, with 4 mM Ca2+ Mus musculus
0.00075
-
 methionine-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mus musculus
0.00125
-
 methionine-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K Mus musculus
0.00152
-
 (S)-3-amino-4-mercapto-butyl sulfonic acid pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K, with 4 mM Ca2+ Mus musculus
0.00157
-
 (4R)-4-amino-5-sulfanylpentanoic acid pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mus musculus
0.00172
-
 (4R)-4-amino-5-sulfanylpentanoic acid pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A Mus musculus
0.002
-
 (S)-3-amino-4-mercapto-butyl sulfonic acid pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A, with 4 mM Ca2+ Mus musculus
0.00322
-
 methionine-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A Mus musculus
0.00514
-
 methionine-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K Mus musculus
0.0065
-
 4-amino-4-phosphonobutyric acid pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A, with 4 mM Ca2+ Mus musculus
0.00751
-
 2,6-diaminohexane-1-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A, with 4 mM Ca2+ Mus musculus
0.00918
-
 methionine-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme, with 4 mM Ca2+ Mus musculus
0.01
-
 (4R)-4-amino-5-sulfanylpentanoic acid pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K Mus musculus
0.0134
-
 2,6-diaminohexane-1-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme, with 4 mM Ca2+ Mus musculus
0.0139
-
 2,6-diaminohexane-1-thiol pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K, with 4 mM Ca2+ Mus musculus

General Information

General Information Comment Organism
malfunction mutagenic replacement of Arg-878 with an alanine or a lysine residue decreases the affinity of the recombinant enzymes for the acidic substrate, alpha-L-glutamyl-beta-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modify the substrate specificity of enzyme APA for the acidic substrate. The mutated enzymes hydrolyse more efficiently basic and neutral substrates, although the addition of Ca2+ partially restores the acidic substrate specificity Mus musculus
additional information residue Arg878 together with Ca2+ is responsible in mouse aminopeptidase A for the substrate specificity for N-terminal acidic amino-acid residues by ensuring the optimal positioning of acidic substrates during catalysis. Docking and molecular dynamics simulations using the crystal structure of human aminopeptidase A complexed with Glu and Ca2+, PDB ID 4KXD, overview Mus musculus
physiological function aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals Mus musculus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.3
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K Mus musculus
3.3
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A Mus musculus
84.3
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme Mus musculus
102
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878A, with 4 mM Ca2+ Mus musculus
502
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged mutant R878K, with 4 mM Ca2+ Mus musculus
2090
-
 alpha-L-glutamyl-beta-naphthylamide pH 7.4, temperature not specified in the publication, recombinant His-tagged wild-type enzyme, with 4 mM Ca2+ Mus musculus