Information on EC 3.4.21.111 - aqualysin 1

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The expected taxonomic range for this enzyme is: Thermus

EC NUMBER
COMMENTARY hide
3.4.21.111
-
RECOMMENDED NAME
GeneOntology No.
aqualysin 1
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
exhibits low specificity towards esters of amino acids with small hydrophobic or aromatic residues at the P1 position
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
cleavage of C-N-linkage
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
88747-68-6
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
casein + H2O
?
show the reaction diagram
chromogenic succinyl-tripeptide-4-nitroanilide + H2O
?
show the reaction diagram
elastin-orcein + H2O
?
show the reaction diagram
Hammarsten casein + H2O
?
show the reaction diagram
N-benzyloxycarbonyl-L-leucyl-4-nitrophenyl ester + H2O
N-benzyloxycarbonyl-L-leucine + 4-nitrophenol
show the reaction diagram
N-succinyl-Ala-Ala-Ala-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Ala + 4-nitroaniline
show the reaction diagram
N-succinyl-Ala-Ala-Phe-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Phe + 4-nitroaniline
show the reaction diagram
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
?
show the reaction diagram
-
-
-
-
?
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Phe + 4-nitroaniline
show the reaction diagram
N-succinyl-Ala-Ala-Val-Ala-4-nitroanilide + H2O
N-succinyl-Ala-Ala-Val-Ala + 4-nitroaniline
show the reaction diagram
-
-
-
?
N-succinyl-Ala-Pro-Ala-4-nitroanilide + H2O
N-succinyl-Ala-Pro-Ala + 4-nitroaniline
show the reaction diagram
-
-
-
?
N-succinyl-Gly-betaAla-Ala-4-nitroanilide + H2O
N-succinyl-Gly-betaAla-Ala + 4-nitroaniline
show the reaction diagram
-
-
-
?
N-succinyl-Gly-Gly-Phe-4-nitroanilide + H2O
N-succinyl-Gly-Gly-Phe + 4-nitroaniline
show the reaction diagram
N-succinyl-Ile-betaAla-Ala-4-nitroanilide + H2O
N-succinyl-Ile-betaAla-Ala + 4-nitroaniline
show the reaction diagram
-
-
-
?
N-succinyl-Ile-Val-Ala-4-nitroanilide + H2O
N-succinyl-Ile-Val-Ala + 4-nitroaniline
show the reaction diagram
N-succinyl-Leu-betaAla-Ala-4-nitroanilide + H2O
N-succinyl-Leu-betaAla-Ala + 4-nitroaniline
show the reaction diagram
-
-
-
?
N-succinyl-Phe-Ala-Ala-4-nitroanilide + H2O
N-succinyl-Phe-Ala-Ala + 4-nitroaniline
show the reaction diagram
N-succinyl-Phe-betaAla-Ala-4-nitroanilide + H2O
N-succinyl-Phe-betaAla-Ala + 4-nitroaniline
show the reaction diagram
-
-
-
?
N-succinyl-Phe-Leu-Ala-4-nitroanilide + H2O
N-succinyl-Phe-Leu-Ala + 4-nitroaniline
show the reaction diagram
N-succinyl-Phe-Nle-Ala-4-nitroanilide + H2O
N-succinyl-Phe-Nle-Ala + 4-nitroaniline
show the reaction diagram
N-succinyl-Phe-Val-Ala-4-nitroanilide + H2O
N-succinyl-Phe-Val-Ala + 4-nitroaniline
show the reaction diagram
N-succinyl-Phe-Val-Leu-4-nitroanilide + H2O
N-succinyl-Phe-Val-Leu + 4-nitroaniline
show the reaction diagram
-
-
-
?
N-succinyl-Phe-Val-Phe-4-nitroanilide + H2O
N-succinyl-Phe-Val-Phe + 4-nitroaniline
show the reaction diagram
-
-
-
?
N-tert-butyloxycarbonyl-Val-Leu-Gly-Arg-4-nitroanilide + H2O
?
show the reaction diagram
-
-
-
?
oxidized insulin chain B + H2O
?
show the reaction diagram
Suc-Ala-Ala-Pro-Phe-p-nitroanilide + H2O
Suc-Ala-Ala-Pro-Phe + p-nitroaniline
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
additional information
?
-
-
requires its propeptide ProA to function as an intramolecular chaperone, N-terminal IMC can inhibit aqualysin activity and form a stable complex with subtilisin BPN'
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Na+
-
binds to low-affinity Ca2+ binding site, binding is essential for thermostabilization
Nd3+
-
stabilizes
Sr2+
-
stabilizes
Tb3+
-
stabilizes
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
-
inhibits S102H/G131H mutant at 0.76 mM, inhibition can be restored by addition of EDTA
DFP
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strongly inhibits
microbial alkaline protease inhibitor
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98% inhibition at 0.001 mg/ml
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N-terminal propeptide of aqualisin I
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potent inhibitor
-
Ni2+
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inhibits S102H/G131H mutant at 0.76 mM, inhibition can be restored by addition of EDTA
Streptomyces subtilisin inhibitor
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Z-Ala-Gly-Phe-CH2Cl
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strongly inhibits
Z-Ala-Gly-PheCH2Cl
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almost complete inhibition at 0.5 mM
Zn2+
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inhibits S102H/G131H mutant at 0.76 mM, inhibition can be restored by addition of EDTA
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.1 - 20
N-succinyl-Ala-Ala-Ala-4-nitroanilide
0.91 - 1.7
N-succinyl-Ala-Ala-Phe-4-nitroanilide
0.49 - 3.06
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
1.6
N-succinyl-Ala-Ala-Val-4-nitroanilide
-
pH 7.4, 40C
0.4 - 0.7
N-succinyl-Ala-Ala-Val-Ala-4-nitroanilide
0.79
N-succinyl-Ala-Pro-Ala-4-nitroanilide
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pH 7.4, 40C
1.6 - 5.3
N-succinyl-Gly-Gly-Phe-4-nitroanilide
0.064 - 1
N-succinyl-Ile-Val-Ala-4-nitroanilide
0.04 - 44
N-succinyl-Phe-Ala-Ala-4-nitroanilide
0.087 - 71
N-succinyl-Phe-Leu-Ala-4-nitroanilide
0.036 - 730
N-succinyl-Phe-Nle-Ala-4-nitroanilide
0.039 - 1200
N-succinyl-Phe-Val-Ala-4-nitroanilide
0.41
N-succinyl-Phe-Val-Leu-4-nitroanilide
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pH 7.4, 40C
2.6 - 2.9
N-tert-butyloxycarbonyl-Val-Leu-Gly-Arg-4-nitroanilide
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.5 - 8.5
N-succinyl-Ala-Ala-Ala-4-nitroanilide
0.009 - 2.5
N-succinyl-Ala-Ala-Phe-4-nitroanilide
0.014 - 296
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
0.047
N-succinyl-Ala-Ala-Val-4-nitroanilide
Thermus aquaticus
-
pH 7.4, 40C
9 - 24
N-succinyl-Ala-Ala-Val-Ala-4-nitroanilide
0.48
N-succinyl-Ala-Pro-Ala-4-nitroanilide
Thermus aquaticus
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pH 7.4, 40C
0.024 - 0.25
N-succinyl-Gly-Gly-Phe-4-nitroanilide
5 - 8.1
N-succinyl-Ile-Val-Ala-4-nitroanilide
0.1 - 11
N-succinyl-Phe-Ala-Ala-4-nitroanilide
3.3 - 3.6
N-succinyl-Phe-Leu-Ala-4-nitroanilide
0.18 - 47400
N-succinyl-Phe-Nle-Ala-4-nitroanilide
0.18 - 4.7
N-succinyl-Phe-Val-Ala-4-nitroanilide
0.069
N-succinyl-Phe-Val-Leu-4-nitroanilide
Thermus aquaticus
-
pH 7.4, 40C
5
N-succinyl-Phe-Val-Phe-4-nitroanilide
Thermus aquaticus
-
pH 7.4, 40C
17 - 42
N-tert-butyloxycarbonyl-Val-Leu-Gly-Arg-4-nitroanilide
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
63.6 - 116
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanilide
953
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
wild type
10 - 11.5
-
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 10.4
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-
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80 - 100
-
wild type enzyme fully active
90
-
wild type and mutant enzymes with lower activity of the mutant enzymes
100
-
P240N and P268T mutant
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 90
temperature-dependence of the activity of the wild-type and mutant enzymes, overview
40 - 100
-
activity curves given for wild type and all mutant enzymes, no differences in activity from 40-60C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9
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pI-value above pH 9-10
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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most activity
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Manually annotated by BRENDA team
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mature protein
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Manually annotated by BRENDA team
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little activity
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
12090
-
ProA peptide, calculated from nucleic acid sequence
14000
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ProA peptide, gel filtration
28350
-
calculated
28500
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SDS-PAGE
53910
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synthesized as a large precursor, calculated
80000
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SDS-PAGE, maltose binding protein-proaqualysin I fusion protein
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure analysis, overview
hanging and sitting drop vapor diffusion method
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70 - 80
75.7
-
transition temperature of the P7I mutant enzyme
83.5
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transition temperature of the P5N mutant enzyme
86.5
-
transition temperature of the P268T mutant enzyme
86.7
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transition temperature of the C194S mutant
86.9
-
transition temperature of the C99S/C194S mutant
89.8
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transition temperature of the C99S mutant
91.8
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transition temperature of the P240N mutant enzyme
94
-
transition temperature of the wild type enzyme
95
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presence of Ca2+, 25% activity after 30 min
additional information
significance of salt bridges in the thermal stability of the enzyme, overview
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
stable against 2 M urea and 2 M guanidine hydrochloride
-
stable to denaturing reagents like 7M urea, 6M guanidine/HCl and 1% SDS
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stable towards denaturing reagents such as 2.5% Tween-20, 4M guanidine HCl or 4M urea
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
38000 Da precursor protein
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disulfide-containing chymotryptic peptides
-
homogeneity
-
ProA peptide
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recombinant enzyme from Escherichia coli cells harboring an expression plasmid using the tac promoter by cation-exchange chromatography, larger scale production by using bacteriophage T7 RNA polymerase/promoter
-
recombinant proteins
-
using hydrophobic chromatography ad cation-exchange-chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
C-terminal deletion mutant, expressed in Escherichia coli
-
DNAa and amino acid sequence determination and analysis, comparisons of primary structures, expression of wild-type and mutant enzymes fused to the maltose binding protein in Escherichia coli stain TG1, subcloning in Escherichia coli strains DH5alpha and MV1184
expressed in Escherichia coli as a maltose biding protein-proaqualysin I fusion protein
-
expression in Escherichia coli
-
expression in Escherichia coli and Thermus thermophilus
-
expression in Pichia pastoris
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mutant with deletion of the C-terminal pro-sequence, expressed in Saccharomyces cerevisiae
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ProA peptide
-
wild type and mutant proteins expressed in Escherichia coli TG1
-
wild-type and C-terminal deletion mutants
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C194S
-
6% of wild type activity at 40C, less thermostable than the wild type enzyme, similar secondary structure as the wild type enzyme
C5A/C8A
-
intracellular distribution similar to wild-type enzyme
C5S/C8S
-
intracellular distribution similar to wild-type enzyme
C99S
-
69% of wild type activity at 40C, almost as stable as the wild type enzyme, similar secondary structure as the wild type enzyme
C99S/C194S
-
3% of wild type activity at 40C, less thermostable than the wild type enzyme, similar secondary structure as the wild type enzyme
D138N
site-sirected mutagenesis
D17N
site-sirected mutagenesis, the mutant is less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis
D183N
site-sirected mutagenesis, the disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant results in a significant reduction in thermal stability, and a massive change in the content of the secondary structure compared to the wild-type enzyme
D212N
site-sirected mutagenesis, thermal stability of D212N is similar to that of the wild-type enzyme at 70C, but it is inactivated rapidly at 80C, the mutant is more prone to unfolding at 80C than the wild-type enzyme
D58N
site-sirected mutagenesis
E237Q
site-sirected mutagenesis, the mutant is less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis. Disruption of a salt bridge in E237Q results in a rapid decrease of activity during incubation at 70C and 80C
G101A
-
catalytic efficiencies for bulky amino acid residues in P2 site such as valine and lucine drastically decreased
G101L
-
reduced catalytic efficiencies for any substrate
G101V
-
catalytic efficiency toward glycine was retained
G131D
-
reduced efficiency for N-succinyl-Phe-Ala-Ala-4-nitroanilide, raised kcat for N-succinyl-Ala-Ala-Ala-4-nitroanilide
G131H
-
reduced efficiency for N-succinyl-Phe-Ala-Ala-4-nitroanilide, raised kcat for N-succinyl-Ala-Ala-Ala-4-nitroanilide
G131K
-
reduced efficiency for N-succinyl-Phe-Ala-Ala-4-nitroanilide, raised kcat for N-succinyl-Ala-Ala-Ala-4-nitroanilide
G262D
site-sirected mutagenesis
G61D
site-sirected mutagenesis
N219S
-
increased catalytic activity
N219T
-
slightly decreased catalytic activity
P240N
-
no effect on the specific activity, almost no effect on the thermostability of the protein
P268T
-
no effect on the specific activity, mutation reduces the thermostability of the protein
P5N
-
no effect on the specific activity, mutation reduces the thermostability of the protein
P7I
-
no effect on the specific activity mutation strongly reduces the thermostability of the protein
S102E
-
reduced efficiency for N-succinyl-Phe-Ala-Ala-4-nitroanilide, raised kcat for N-succinyl-Ala-Ala-Ala-4-nitroanilide
S102H
-
reduced efficiency for N-succinyl-Phe-Ala-Ala-4-nitroanilide, raised kcat for N-succinyl-Ala-Ala-Ala-4-nitroanilide
S102H/G131H
-
enzyme can be inhibited by metal ions
S102K
-
reduced efficiency for N-succinyl-Phe-Ala-Ala-4-nitroanilide, raised kcat for N-succinyl-Ala-Ala-Ala-4-nitroanilide
S22A
-
inactive mutant
S277D
site-sirected mutagenesis
C194S
-
6% of wild type activity at 40C, less thermostable than the wild type enzyme, similar secondary structure as the wild type enzyme
-
C5A/C8A
-
intracellular distribution similar to wild-type enzyme
-
C5S/C8S
-
intracellular distribution similar to wild-type enzyme
-
C99S
-
69% of wild type activity at 40C, almost as stable as the wild type enzyme, similar secondary structure as the wild type enzyme
-
C99S/C194S
-
3% of wild type activity at 40C, less thermostable than the wild type enzyme, similar secondary structure as the wild type enzyme
-
D138N
-
site-sirected mutagenesis
-
D17N
-
site-sirected mutagenesis, the mutant is less thermostable than the wild-type enzyme, although this may be partially due to increased autolysis
-
D183N
-
site-sirected mutagenesis, the disruption of a salt bridge common to proteinase K subfamily enzymes in the D183N mutant results in a significant reduction in thermal stability, and a massive change in the content of the secondary structure compared to the wild-type enzyme
-
D58N
-
site-sirected mutagenesis
-
P240N
-
no effect on the specific activity, almost no effect on the thermostability of the protein
-
P268T
-
no effect on the specific activity, mutation reduces the thermostability of the protein
-
P5N
-
no effect on the specific activity, mutation reduces the thermostability of the protein
-
P7I
-
no effect on the specific activity mutation strongly reduces the thermostability of the protein
-
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
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a maltose biding protein (MBP)-fused proaqualysin I expression plasmid is developed in which MBP is attached to the N-terminus of proaqualysin I. MBP appears effectively to suppress the folding-promoting activity of the N-terminal propeptide when the bacteria are grown at 30C, leading to a massive accumulation of fusion aqualysin I precursor. The precursor is converted efficiently to mature aqualysin I by heat treatment at 70C. By analyzing the product it is confirmed that aqualysin I is initially expressed as a whole fusion protein and then processed autocatalytically
additional information