Information on EC 3.4.24.B16 - protease lasA

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The expected taxonomic range for this enzyme is: Pseudomonas aeruginosa

EC NUMBER
COMMENTARY hide
3.4.24.B16
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
protease lasA
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
proteolytic degradation of proteins
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
66554-76-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain FRD1
SwissProt
Manually annotated by BRENDA team
strain FRD2128(pJKGT107)
-
-
Manually annotated by BRENDA team
strain FRD740
-
-
Manually annotated by BRENDA team
strain PA01
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-(dimethylamino)azobenzene-4'-sulfonyl chloride-Leu-Gly-Gly-Gly-Ala-5-(2-aminoethylamino)-1-naphthalenesulfonic acid + H2O
?
show the reaction diagram
beta-casein + H2O
beta-casein + 30 amino acid residues fragment
show the reaction diagram
-
only 1 cleavage site: in the sequence NKKIEKFQphosphorylated-S between Lys and Ile, serine protease activity
-
?
Elastin + H2O
?
show the reaction diagram
LGGGA + H2O
Lys-Gly-Gly + Gly-Ala
show the reaction diagram
-
best substrate
-
-
?
protein + H2O
peptides
show the reaction diagram
succinyl-Gly-Gly-Leu-4-nitroanilide + H2O
Leu-4-nitroanilide + succinyl-Gly-Gly
show the reaction diagram
succinyl-Gly-Gly-Phe-4-nitroanilide + H2O
Phe-4 nitroanilide + succinyl-Gly-Gly
show the reaction diagram
tropoelastin + H2O
?
show the reaction diagram
-
preferentially cleave at Gly-Gly-Ala
-
-
?
additional information
?
-
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
Elastin + H2O
?
show the reaction diagram
protein + H2O
peptides
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Zinc
-
the active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules, crystallization data
Zn2+
-
zinc-metalloprotease
additional information
-
enzyme is no metalloprotease
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
dithiothreitol
-
inhibition of the staphylolytic activity
N-tosyl-L-lysine chloromethyl ketone
-
-
Nalpha-p-tosyl-L-lysine chloromethyl ketone
-
inhibition of the staphylolytic activity
Phenylmethylsulfonylfluoride
-
-
phosphoramidon
-
inhibition of the staphylolytic activity
tetraethylene pentamine
-
-
Tetraethylenepentamine
-
inhibition of the staphylolytic activity
Trypsin
cleaves the enzyme
-
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
elastase enhances staphylolytic activity of LasA
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1
4-(dimethylamino)azobenzene-4'-sulfonyl-Leu-Gly-Gly-Gly-Ala-5-(2-aminoethylamino)-1-naphthalenesulfonic acid
-
in 5 mM Tris-HCl buffer, pH 8.5
11.2
succinyl-Gly-Gly-Leu-4-nitroanilide
-
-
0.46
succinyl-Gly-Gly-Phe-4-nitroanilide
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
15.2
4-(dimethylamino)azobenzene-4'-sulfonyl-Leu-Gly-Gly-Gly-Ala-5-(2-aminoethylamino)-1-naphthalenesulfonic acid
Pseudomonas aeruginosa
-
in 5 mM Tris-HCl buffer, pH 8.5
3.55
succinyl-Gly-Gly-Leu-4-nitroanilide
Pseudomonas aeruginosa
-
-
11.8
succinyl-Gly-Gly-Phe-4-nitroanilide
Pseudomonas aeruginosa
-
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.5
-
above, isoelectric focussing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
inactive precursor
Manually annotated by BRENDA team
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inactive precursor
-
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3298
-
preproenzyme, calculation from amino-acid sequence
19000
-
x * 19000, SDS-PAGE
19963
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x * 19963, electrospray mass spectrometry, mature protein
19970
-
calculation from amino-acid sequence
22000
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x * 22000, SDS-PAGE
22320
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N-terminal propeptide, calculation from amino-acid sequence
45582
x * 20000, LasA, SDS-PAGE, x * 42000, proLasA, SDS-PAGE, x * 45582, pre-proLasA, DNA sequence determination
98000
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SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structures of active LasA as a complex with tartrate and in the uncomplexed form, to 1.28 A resolution. The overall fold resembles that of the other M23 family members, the LasA active site is less constricted and utilizes a different set of metal ligands. The active site of uncomplexed LasA contains a five-coordinate zinc ion with trigonal bipyramidal geometry and two metal-bound water molecules. Manual docking study of the pentapeptide Gly-Gly-Phe-Gly-Gly in the active site so that the carbonyl oxygen of the scissile peptide occupies the approximate position of Tyr151-bound tartrate oxygen O1 and the P2 and P1 glycines follow the path of the tartrate carbon skeleton
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-80°C, HEPES buffer, pH 7.8
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by chromatography
-
DEAE-Sepharose Fast Flow chromatography
-
from culture supernatant
-
from the culture medium
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from the culture supernatant
to homogeneity by chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA sequence determination and analysis, overexpression of gene lasA in Escherichia coli strain JM109 in form of the pre-proLasA precursor protein
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the production of LasA is reduced by Terminalia catappa active extract (fraction 12) in a concentration dependent manner with about 50% reduction at 0.0625 mg/ml
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
G115A
-
slightly reduced activity
H120A
oligonucleotide-directed mutagenesis, formation of the lasA5 allele, processing to a 20 kDa LasA-H120A protein and secretion to the medium occurs, but the mutant enzyme has no staphylolytic activity
H94A
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mutation is moderately unstable and allows the production of a LasA protein with low enzymatic activity
I101A
-
mutation is moderately unstable and allows the production of a LasA protein with low enzymatic activity
L92A
-
mutation results in highly unstable protein that is susceptible to proteolytic degradation
N102A
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mutation is moderately unstable and allows the production of a LasA protein with low enzymatic activity
W95A
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mutation results in highly unstable protein that is susceptible to proteolytic degradation
H120A
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oligonucleotide-directed mutagenesis, formation of the lasA5 allele, processing to a 20 kDa LasA-H120A protein and secretion to the medium occurs, but the mutant enzyme has no staphylolytic activity
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
degradation
medicine
additional information
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cell lysis of Staphylococcus aureus, Micrococcus radiodurans and Gaffkia tetragena