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Literature summary extracted from

  • Webster, C.I.; Burrell, M.; Olsson, L.L.; Fowler, S.B.; Digby, S.; Sandercock, A.; Snijder, A.; Tebbe, J.; Haupts, U.; Grudzinska, J.; Jermutus, L.; Andersson, C.
    Engineering neprilysin activity and specificity to create a novel therapeutic for Alzheimers disease (2014), PLoS ONE, 9, e104001.
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
3.4.24.11 medicine engineered neprilysin can be a therapeutic for Alzheimer's disease due to its ability to degrade the peptide amyloid beta Homo sapiens

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.4.24.11 recombinant expression of His10-tagged wild-type and mutant enzymes in CHO suspension cells, recombinant expression of HA-tagged wild-type and mutant enzymes in Saccharomyces cerevisiae strain YMR307w Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.4.24.11 purified recombinant enzyme mutant G399V/G714K in complex with inhibitor phorphoramidon, hanging drop vapor diffusion method, mixing of 0.001 ml of 10 mg/ml protein in 25 mM Tris-HCl, pH 7.0, 150 mM NaCl and 2 mM MgCl2 with 2 mM phosphoramidon, 0.001 ml of reservoir solution containing 100 mM HEPES, pH 7.0, 22% w/v PEG3350 and 200 mM NaCl, at 20°C, 2 weeks, rod shaped crystals, improvement by streak seeding, X-ray diffraction structure determination and analysis at 2.15 A resolution, modeling Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
3.4.24.11 G399V site-directed mutagenesis, the mutant shows increased catalytic efficiency on Ab1-40 with 6fold increased catalytic efficiency compared to the wild-type enzyme. The G399V mutation also significantly reduces the catalytic efficiency on angiotensin, bradykinin and neurotensin compared to the wild-type enzyme Homo sapiens
3.4.24.11 G399V/G714K site-directed mutagenesis, the enzyme variant displays an approximately 20fold improved activity on amyloid beta 1-40 and up to a 3200fold reduction in activity on other peptides, and the mutant enzyme produces a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme Homo sapiens
3.4.24.11 G714K site-directed mutagenesis, the mutant shows increased catalytic efficiency on Ab1-40 with 6fold increased catalytic efficiency compared to the wild-type enzyme Homo sapiens
3.4.24.11 additional information generation of a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta peptide as a potential therapeutic for Alzheimer's disease Homo sapiens

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.4.24.11 phosphoramidon
-
Homo sapiens
3.4.24.11 thiorphan
-
Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.4.24.11 additional information
-
additional information Michaelis-Menten profiles and steady-state kinetic parameters for cleavage of amyloid beta peptide1-40 and off-target peptide by wild-type and mutant enzymes, overview Homo sapiens
3.4.24.11 0.0062
-
Arg-vasopressin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.0074
-
somatostatin 14 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.0086
-
angiotensin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.0093
-
Neurokinin A wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.013
-
gamma-endorphin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.014
-
amyloid beta peptide1-40 mutant G399V/G714K, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.016
-
Substance P wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.016
-
neurotensin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.02
-
nociceptin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.022
-
neurokinin B wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.024
-
GLP-1 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.024
-
alpha-endorphin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.025
-
glucagon wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.031
-
somatostatin 28 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.032
-
bradykinin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.042
-
amyloid beta peptide1-40 mutant G714K, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.053
-
endothelin-1 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.055
-
amyloid beta peptide1-40 mutant G399V, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.104
-
amyloid beta peptide1-40 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.15
-
gastri-releasing peptide wild-type enzyme, pH and temperature not specified in the publication Homo sapiens

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
3.4.24.11 membrane a transmembrane zinc metallopeptidase Homo sapiens 16020
-

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.4.24.11 Zn2+ a transmembrane zinc metallopeptidase Homo sapiens

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.4.24.11 82000 82410 recombinant enzyme, gel filtration Homo sapiens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.4.24.11 amyloid beta peptide + H2O Homo sapiens degradation ?
-
?
3.4.24.11 amyloid beta peptide1-40 + H2O Homo sapiens degradation ?
-
?
3.4.24.11 amyloid beta peptide1-42 + H2O Homo sapiens degradation ?
-
?
3.4.24.11 additional information Homo sapiens the enzyme degrades a wide range of peptide substrates, physiologically relevant peptides are such as enkephalins, tachykinins, and natriuretic peptides ?
-
?

Organism

EC Number Organism UniProt Comment Textmining
3.4.24.11 Homo sapiens P08473
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.4.24.11 recombinant His10-tagged wild-type and mutant enzymes from CHO suspension cells by nickel affinity chromatography, recombinant HA-tagged wild-type and mutant enzymes from Saccharomyces cerevisiae strain YMR307w by immunoaffinity chromatography Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.4.24.11 alpha-endorphin + H2O
-
Homo sapiens ?
-
?
3.4.24.11 amyloid beta peptide + H2O
-
Homo sapiens ?
-
?
3.4.24.11 amyloid beta peptide + H2O degradation Homo sapiens ?
-
?
3.4.24.11 amyloid beta peptide1-40 + H2O degradation Homo sapiens ?
-
?
3.4.24.11 amyloid beta peptide1-40 + H2O the wild-type enzyme cleaves Ab1-40 predominantly at Lys16-Leu17, Leu17-Val18 and Phe19-Phe20. The mutant G399V/G714K cleaves preferentially at Phe20-Ala21 Homo sapiens amyloid beta peptide Asp1-Lys16 + amyloid beta peptide Asp1-Leu17 + amyloid beta peptide Asp1-Phe19 main cleavage fragments after 60 min with the wild-type enzyme. After 360 min these fragments are degraded further, accompanied by the appearance of Val12-Leu17 and Tyr10-Leu17 fragments ?
3.4.24.11 amyloid beta peptide1-42 + H2O
-
Homo sapiens ?
-
?
3.4.24.11 amyloid beta peptide1-42 + H2O degradation Homo sapiens ?
-
?
3.4.24.11 angiotensin + H2O
-
Homo sapiens ?
-
?
3.4.24.11 Arg-vasopressin + H2O
-
Homo sapiens ?
-
?
3.4.24.11 bradykinin + H2O
-
Homo sapiens ?
-
?
3.4.24.11 endothelin-1 + H2O
-
Homo sapiens ?
-
?
3.4.24.11 gamma-endorphin + H2O
-
Homo sapiens ?
-
?
3.4.24.11 gastri-releasing peptide + H2O
-
Homo sapiens ?
-
?
3.4.24.11 GLP-1 + H2O
-
Homo sapiens ?
-
?
3.4.24.11 Glucagon + H2O
-
Homo sapiens ?
-
?
3.4.24.11 additional information the enzyme degrades a wide range of peptide substrates, physiologically relevant peptides are such as enkephalins, tachykinins, and natriuretic peptides Homo sapiens ?
-
?
3.4.24.11 Neurokinin A + H2O
-
Homo sapiens ?
-
?
3.4.24.11 neurokinin B + H2O
-
Homo sapiens ?
-
?
3.4.24.11 neurotensin + H2O
-
Homo sapiens ?
-
?
3.4.24.11 nociceptin + H2O
-
Homo sapiens ?
-
?
3.4.24.11 somatostatin 14 + H2O
-
Homo sapiens ?
-
?
3.4.24.11 somatostatin 28 + H2O
-
Homo sapiens ?
-
?
3.4.24.11 Substance P + H2O
-
Homo sapiens ?
-
?

Synonyms

EC Number Synonyms Comment Organism
3.4.24.11 NEP
-
Homo sapiens
3.4.24.11 neutral endopeptidase
-
Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.4.24.11 0.68
-
endothelin-1 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 0.83
-
amyloid beta peptide1-40 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 1.9
-
amyloid beta peptide1-40 mutant G714K, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 2.1
-
amyloid beta peptide1-40 mutant G399V/G714K, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 2.7
-
amyloid beta peptide1-40 mutant G399V, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 2.8
-
Arg-vasopressin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 3.3
-
GLP-1 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 4.5
-
glucagon wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 4.8
-
gamma-endorphin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 8.2
-
angiotensin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 8.9
-
alpha-endorphin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 9.1
-
somatostatin 28 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 11
-
nociceptin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 13
-
neurotensin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 16
-
bradykinin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 17
-
Neurokinin A wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 18
-
Substance P wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 20
-
neurokinin B wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 26
-
gastri-releasing peptide wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 32
-
somatostatin 14 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
3.4.24.11 0.00001
-
wild-type enzyme, pH and temperature not specified in the publication Homo sapiens phosphoramidon
3.4.24.11 0.00001
-
below, wild-type enzyme, pH and temperature not specified in the publication Homo sapiens thiorphan
3.4.24.11 0.000075
-
enzyme mutant G714K, pH and temperature not specified in the publication Homo sapiens phosphoramidon
3.4.24.11 0.0044
-
enzyme mutant G399V, pH and temperature not specified in the publication Homo sapiens phosphoramidon
3.4.24.11 0.021
-
enzyme mutant G399V/G714K, pH and temperature not specified in the publication Homo sapiens phosphoramidon
3.4.24.11 0.077
-
enzyme mutant G399V/G714K, pH and temperature not specified in the publication Homo sapiens thiorphan

General Information

EC Number General Information Comment Organism
3.4.24.11 evolution the enzyme harbors an ectodomain, which contains the catalytic site and belongs to the M13 family of peptidases/proteases Homo sapiens
3.4.24.11 additional information the enzyme is composed of an ectodomain, which contains the catalytic site, a transmembrane domain and a short intracellular domain. The structure of the ectodomain is composed of two largely alpha-helical domains that are arranged to form a central spherical water-filled core that contains the active site of the enzyme Homo sapiens
3.4.24.11 physiological function the enzyme degrades a number of physiological peptides that are involved in processes such as blood pressure regulation and nociception Homo sapiens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.4.24.11 8
-
amyloid beta peptide1-40 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 13
-
endothelin-1 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 45
-
amyloid beta peptide1-40 mutant G714K, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 49
-
amyloid beta peptide1-40 mutant G399V, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 140
-
GLP-1 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 150
-
amyloid beta peptide1-40 mutant G399V/G714K, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 160
-
somatostatin 28 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 170
-
gastri-releasing peptide wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 180
-
glucagon wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 380
-
gamma-endorphin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 390
-
alpha-endorphin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 450
-
Arg-vasopressin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 480
-
bradykinin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 570
-
nociceptin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 820
-
neurotensin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 920
-
neurokinin B wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 970
-
angiotensin wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 1100
-
Substance P wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 1700
-
Neurokinin A wild-type enzyme, pH and temperature not specified in the publication Homo sapiens
3.4.24.11 4400
-
somatostatin 14 wild-type enzyme, pH and temperature not specified in the publication Homo sapiens