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Information on EC 3.4.11.10 - bacterial leucyl aminopeptidase and Organism(s) Bacillus subtilis and UniProt Accession P25152

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EC Tree
     3 Hydrolases
         3.4 Acting on peptide bonds (peptidases)
             3.4.11 Aminopeptidases
                3.4.11.10 bacterial leucyl aminopeptidase
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Bacillus subtilis
UNIPROT: P25152 not found.
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The taxonomic range for the selected organisms is: Bacillus subtilis
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Reaction Schemes
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Release of an N-terminal amino acid, preferentially leucine, but not glutamic or aspartic acids.
release of an N-terminal amino acid, preferentially leucine, but not glutamic or aspartic acids
Synonyms
cgase, ap-ii, cysteinylglycinase, fglap, hpm17ap, lapii, mtlap, m17 aminopeptidase, thermostable leucine aminopeptidase, rlap55, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Bsu aminopeptidase
-
double-zinc aminopeptidase
-
Aeromonas proteolytica aminopeptidase
-
-
-
-
extracellular aminopeptidase
-
-
leucine aminopeptidase
ribosomal-bound aminopeptidase
-
-
-
-
additional information
see also EC 3.4.11.1
CAS REGISTRY NUMBER
COMMENTARY hide
37288-67-8
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
L-Ala-4-nitroanilide + H2O
L-Ala + 4-nitroaniline
show the reaction diagram
low activity
-
-
?
L-Arg-4-nitroanilide + H2O
L-Arg + 4-nitroaniline
show the reaction diagram
best substrate
-
-
?
L-Leu-4-nitroanilide + H2O
L-Leu + 4-nitroaniline
show the reaction diagram
-
-
-
?
L-Lys-4-nitroanilide + H2O
L-Lys + 4-nitroaniline
show the reaction diagram
-
-
-
?
L-Val-4-nitroanilide + H2O
L-Val + 4-nitroaniline
show the reaction diagram
low activity
-
-
?
Arg-4-nitroanilide + H2O
Arg + 4-nitroaniline
show the reaction diagram
74.5% activity compared to Leu-4-nitroanilide
-
-
?
L-Leu-4-nitroanilide + H2O
L-Leu + 4-nitroaniline
show the reaction diagram
-
-
-
-
?
Leu-4-nitroanilide + H2O
Leu + 4-nitroaniline
show the reaction diagram
-
-
-
?
leucine-4-nitroaniline + H2O
?
show the reaction diagram
-
enzyme also shows activity towards Arg-4-nitroaniline, Lys-4-nitroaniline and Met-4-nitroaniline
-
-
?
Lys-4-nitroanilide + H2O
Lys + 4-nitroaniline
show the reaction diagram
20% activity compared to Leu-4-nitroanilide
-
-
?
Met-4-nitroanilide + H2O
Met + 4-nitroaniline
show the reaction diagram
8.2% activity compared to Leu-4-nitroanilide
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zn2+
zinc-metalloaminopeptidase, 2 Zn2+ per enzyme molecule
Cu2+
-
weakly inhibits activity
Ni2+
-
inhibits activity
Zn2+
-
dinuclear zinc active center
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
complete inhibition, reversible by Zn2+
2-mercaptoethanol
strong inhibition
Ca2+
68.4% inhibition at 1 mM
Cu2+
slight inhibition at 1 mM
DL-dithiothreitol
-
-
DTT
complete inhibition at 1 mM, 50°C, 1 h
Mg2+
slight inhibition at 1 mM
Mn2+
slight inhibition at 1 mM
Ni2+
92.5% inhibition at 1 mM
PMSF
slight inhibition
Zn2+
slight inhibition at 1 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
10
L-Ala-4-nitroanilide
pH 8.0, 30°C
0.6
L-Arg-4-nitroanilide
pH 8.0, 30°C
3.6
L-Leu-4-nitroanilide
pH 8.0, 30°C
0.85
L-Lys-4-nitroanilide
pH 8.0, 30°C
19
L-Val-4-nitroanilide
pH 8.0, 30°C
0.97
Leu-4-nitroanilide
pH 8.5, 50°C, recombinant enzyme
0.97
leucine-4-nitroaniline
-
pH 8.5, 60°C
additional information
additional information
kinetics
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.37
L-Ala-4-nitroanilide
pH 8.0, 30°C
34
L-Arg-4-nitroanilide
pH 8.0, 30°C
55
L-Leu-4-nitroanilide
pH 8.0, 30°C
10
L-Lys-4-nitroanilide
pH 8.0, 30°C
0.43
L-Val-4-nitroanilide
pH 8.0, 30°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
64
purified recombinant enzyme
132.5
purified native enzyme, pH 8.5, 50°C, substrate Leu-4-nitroanilide
240.48
purified recombinant enzyme, pH 8.5, 50°C, substrate Leu-4-nitroanilide
additional information
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
the enzyme BsuAP is not just thermostable but also thermophilic in character and activity
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
46425
-
1 * 46425, sequence calculation
50000
-
SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
-
1 * 46425, sequence calculation
additional information
-
three-dimensional structure analysis
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme with or without Zn2+, hanging drop vapour diffusion method, method screening, 0.006 ml protein solution against 1 ml reservoir solution, which contains2.0-2.5 M ammonium sulfate, 14-60 days, X-ray diffraction structure determination and analysis at 2.2-2.5 A resolution, multiwavelength anomalous diffraction
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8 - 9
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
20 min, purified recombinant enzyme, stable
30 - 60
-
recombinant protein
30 - 70
purified recombinant enzyme, stable at 30-60°C. The enzyme retains 63.5% activity after incubation at 70°C for 1 h, and 75% activity after 3 h at 30-60°C
80
-
20 min, purified recombinant enzyme, stable
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme 5fold from Escherichia coli by ion exchange chromatography
recombinant enzyme from Escherichia coli by ion exchange chromatography
-
recombinant extracellular enzyme 8.4fold from Pichia pastoris strain GS115 culture supernatant by dialysis, anion exchange chromatography, ultrafiltration, and gel filtration
using ion-exchange chromatography, ultrafiltration and gel filtration
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene ywad, overexpression in Escherichia coli
expressed in Pichia pastoris
-
overexpression in Escherichia coli
-
recombinant expression in Pichia pastoris strain GS115, the enzyme is secreted, the recombinant enzyme level is 7.1 times that of wild-type Bacillus subtilis strain Zj016, subcloning in Escherichia coli strain JM109
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
evaluation of a single-stage MS-based technique for amino acid sequencing involving partial, heterogenous digestion of a peptide by a processive, non-specific, thermotropic Bacillus subtilis-derived aminopeptidase (BsuAP), which allows single-shot sequencing to be carried out through simultaneous accumulation, and detection of subpopulations of peptides of progressively reducing length
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Reiland, V.; Fundoiano-Hershcovitz, Y.; Golan, G.; Gilboa, R.; Shoham, Y.; Shoham, G.
Preliminary crystallographic characterization of BSAP, an extracellular aminopeptidase from Bacillus subtilis
Acta Crystallogr. Sect. D
60
2371-2376
2004
Bacillus subtilis
Manually annotated by BRENDA team
Fundoiano-Hershcovitz, Y.; Rabinovitch, L.; Shulami, S.; Reiland, V.; Shoham, G.; Shoham, Y.
The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase
FEMS Microbiol. Lett.
243
157-163
2005
Bacillus subtilis (P25152)
Manually annotated by BRENDA team
Xi, H.; Tian, Y.; Zhou, N.; Zhou, Z.; Shen, W.
Characterization of an N-glycosylated Bacillus subtilis leucine aminopeptidase expressed in Pichia pastoris
J. Basic Microbiol.
55
236-246
2015
Bacillus subtilis
Manually annotated by BRENDA team
Kishor, N.; Guptasarma, P.
Direct N-terminal sequencing of polypeptides using a thermostable bacterial aminopeptidase and MALDI-TOF mass spectrometry
Anal. Biochem.
488
6-8
2015
Bacillus subtilis (P25152), Bacillus subtilis 168 (P25152)
Manually annotated by BRENDA team
Xi, H.; Tian, Y.; Zhou, N.; Zhou, Z.; Shen, W.
Characterization of an N-glycosylated Bacillus subtilis leucine aminopeptidase expressed in Pichia pastoris
J. Basic Microbiol.
55
236-246
2015
Bacillus subtilis (O32106), Bacillus subtilis Zj016 (O32106)
Manually annotated by BRENDA team