Literature summary for 3.4.24.11 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.

Search for more literature for 3.4.24.11

Application

Application	Comment	Organism
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)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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
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
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
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

Inhibitors	Comment	Organism	Structure
phosphoramidon	-	Homo sapiens
thiorphan	-	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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
0.0062	-	Arg-vasopressin	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.0074	-	somatostatin 14	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.0086	-	angiotensin	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.0093	-	Neurokinin A	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.013	-	gamma-endorphin	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.014	-	amyloid beta peptide1-40	mutant G399V/G714K, pH and temperature not specified in the publication	Homo sapiens
0.016	-	Substance P	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.016	-	neurotensin	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.02	-	nociceptin	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.022	-	neurokinin B	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.024	-	GLP-1	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.024	-	alpha-endorphin	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.025	-	glucagon	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.031	-	somatostatin 28	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.032	-	bradykinin	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.042	-	amyloid beta peptide1-40	mutant G714K, pH and temperature not specified in the publication	Homo sapiens
0.053	-	endothelin-1	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.055	-	amyloid beta peptide1-40	mutant G399V, pH and temperature not specified in the publication	Homo sapiens
0.104	-	amyloid beta peptide1-40	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens
0.15	-	gastri-releasing peptide	wild-type enzyme, pH and temperature not specified in the publication	Homo sapiens

Localization

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

Metals/Ions

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

Molecular Weight [Da]

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

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
amyloid beta peptide + H2O	Homo sapiens	degradation	?	-	?
amyloid beta peptide1-40 + H2O	Homo sapiens	degradation	?	-	?
amyloid beta peptide1-42 + H2O	Homo sapiens	degradation	?	-	?
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

Organism	UniProt	Comment	Textmining
Homo sapiens	P08473	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
alpha-endorphin + H2O	-	Homo sapiens	?	-	?
amyloid beta peptide + H2O	-	Homo sapiens	?	-	?
amyloid beta peptide + H2O	degradation	Homo sapiens	?	-	?
amyloid beta peptide1-40 + H2O	degradation	Homo sapiens	?	-	?
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	?
amyloid beta peptide1-42 + H2O	-	Homo sapiens	?	-	?
amyloid beta peptide1-42 + H2O	degradation	Homo sapiens	?	-	?
angiotensin + H2O	-	Homo sapiens	?	-	?
Arg-vasopressin + H2O	-	Homo sapiens	?	-	?
bradykinin + H2O	-	Homo sapiens	?	-	?
endothelin-1 + H2O	-	Homo sapiens	?	-	?
gamma-endorphin + H2O	-	Homo sapiens	?	-	?
gastri-releasing peptide + H2O	-	Homo sapiens	?	-	?
GLP-1 + H2O	-	Homo sapiens	?	-	?
Glucagon + H2O	-	Homo sapiens	?	-	?
additional information	the enzyme degrades a wide range of peptide substrates, physiologically relevant peptides are such as enkephalins, tachykinins, and natriuretic peptides	Homo sapiens	?	-	?
Neurokinin A + H2O	-	Homo sapiens	?	-	?
neurokinin B + H2O	-	Homo sapiens	?	-	?
neurotensin + H2O	-	Homo sapiens	?	-	?
nociceptin + H2O	-	Homo sapiens	?	-	?
somatostatin 14 + H2O	-	Homo sapiens	?	-	?
somatostatin 28 + H2O	-	Homo sapiens	?	-	?
Substance P + H2O	-	Homo sapiens	?	-	?

Synonyms

Synonyms	Comment	Organism
NEP	-	Homo sapiens
neutral endopeptidase	-	Homo sapiens

Turnover Number [1/s]

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

IC50 Value

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

General Information

General Information	Comment	Organism
evolution	the enzyme harbors an ectodomain, which contains the catalytic site and belongs to the M13 family of peptidases/proteases	Homo sapiens
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
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]

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