Information on EC 3.4.22.48 - staphopain

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The expected taxonomic range for this enzyme is: Staphylococcus

EC NUMBER
COMMENTARY hide
3.4.22.48
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RECOMMENDED NAME
GeneOntology No.
staphopain
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
broad endopeptidase action on proteins including elastin, but rather limited hydrolysis of small-molecule substrates. Assays are conveniently made with hemoglobin, casein or Z-Phe-Arg-NHMec as substrate
show the reaction diagram
CAS REGISTRY NUMBER
COMMENTARY hide
347841-89-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 8325-4 including isogenic knockout mutants in the 8325-4 genetic background
SwissProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
strain BC10
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Manually annotated by BRENDA team
strain CH-91
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Manually annotated by BRENDA team
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-
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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
2-aminobenzoyl-Ile-Ala-Ala-Gly-5-amino-2-nitrobenzoylamide + H2O
?
show the reaction diagram
-
-
-
-
?
2-aminobenzoyl-Ile-Ala-Lys-Asp-5-amino-2-nitrobenzoylamide + H2O
?
show the reaction diagram
-
-
-
-
?
2-aminobenzoyl-Phe-Gly-Ala-Lys-5-amino-2-nitrobenzoylamide + H2O
?
show the reaction diagram
-
-
-
-
?
5-carboxyfluorescein-Lys-Lys-Ala-Ala-Glu-Ala-Ser-Lys-(QXL520)-OH + H2O
?
show the reaction diagram
Abz-Glu-Ala-Leu-Gly-Thr-Ser-Pro-Arg-Lys(Dnp)-Asp + H2O
?
show the reaction diagram
-
-
-
-
?
Abz-Glu-Gly-Ile-Gly-Thr-Ser-Arg-Pro-Lys(Dnp)-Asp + H2O
?
show the reaction diagram
-
-
-
-
?
alcohol dehydrogenase + H2O
alcohol dehydrogenase proteolytically cleaved into peptide fragments
show the reaction diagram
alpha-1-antitrypsin + H2O
alpha-1-antitrypsin proteolytically cleaved into peptide fragments
show the reaction diagram
-
limited proteolysis
-
-
?
alpha1-proteinase inhibitor + H2O
?
show the reaction diagram
-
human protein, inactivation by SspA
-
-
?
benzyl-Tyr-OEt + H2O
?
show the reaction diagram
-
-
-
-
?
Bz-Pro-Phe-Arg-4-nitroanilide + H2O
?
show the reaction diagram
Bz-Pro-Phe-Arg-4-nitroanilide + H2O
Bz-Pro-Phe-Arg + 4-nitroaniline
show the reaction diagram
casein + H2O
casein proteolytically cleaved into peptide fragments
show the reaction diagram
Collagen + H2O
?
show the reaction diagram
-
-
-
-
?
CXCR2 + H2O
?
show the reaction diagram
cystatin C + H2O
?
show the reaction diagram
cystatin D + H2O
?
show the reaction diagram
elastin + H2O
elastin proteolytically cleaved into peptide fragments
show the reaction diagram
-
insoluble substrate
-
-
?
elastin agar + H2O
?
show the reaction diagram
fibrinogen A alpha chain + H2O
?
show the reaction diagram
Gelatin + H2O
?
show the reaction diagram
-
staphopain B
-
-
?
hemoglobin + H2O
hemoglobin proteolytically cleaved into peptide fragments
show the reaction diagram
HMW-kininogen + H2O
HMW-kininogen proteolytically cleaved into peptide fragments
show the reaction diagram
-
limited proteolysis
-
-
?
integrin CD11b + H2O
?
show the reaction diagram
Kininogen + H2O
Kinin
show the reaction diagram
milk agar + H2O
?
show the reaction diagram
N-benzyloxycarbonyl-Phe-Leu-Glu-NH-p-nitroanilide + H2O
N-benzyloxycarbonyl-Phe-Leu-Glu + p-nitroaniline
show the reaction diagram
-
-
-
-
?
N-Suc-Gly-Phe-Gly-p-nitroanilide + H2O
N-Suc-Gly-Phe-Gly + p-nitroaniline
show the reaction diagram
peptide + H2O
amino acids
show the reaction diagram
peptide + H2O
peptide proteolytically cleaved into fragments or single amino acids
show the reaction diagram
protein + H2O
peptide fragments
show the reaction diagram
protein + H2O
protein proteolytically cleaved into peptide fragments
show the reaction diagram
Z-Phe-Leu-Glu-p-nitroanilide + H2O
Z-Phe-Leu-Glu + 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
CXCR2 + H2O
?
show the reaction diagram
cystatin C + H2O
?
show the reaction diagram
cystatin D + H2O
?
show the reaction diagram
integrin CD11b + H2O
?
show the reaction diagram
Kininogen + H2O
Kinin
show the reaction diagram
-
activation of human protein by SspA, kinin generation is responsible for infection associated pain and endema
-
-
?
peptide + H2O
peptide proteolytically cleaved into fragments or single amino acids
show the reaction diagram
protein + H2O
protein proteolytically cleaved into peptide fragments
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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not affected by changes in ionic strength by changing NaCl concentration
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
alpha2-Macroglobulin
E64
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ScpA-inhibitor binding structure
L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane
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E-64, irreversible inhibitor
p-hydroxymercuribenzoate
-
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phosphorylated cystatin alpha
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from rat skin
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squamous cell carcinoma antigen 1
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the high association rate constant (kass) for inhibitory complex formation (19000 M/s for staphopain A interaction with SCCA1) suggests that squamous cell carcinoma antigen 1 (SCCA1) can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by Staphylococcu aureus; the high association rate constant (kass) for inhibitory complex formation (58000 M/s for staphopain A interaction with SCCA1) suggests that squamous cell carcinoma antigen 1 (SCCA1) can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by Staphylococcu aureus
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staphostatin
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staphostatin A
staphostatin B
staphostatins
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co-expression of staphopain and inhibitor staphostatin from the same operon, regulatory effect; endogenous proteins that specifically inhibit staphopain; formation of tight and stable non-covalent complexes
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T-Kininogen
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from rat
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additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
reducing thiol compounds
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dependent on
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0076 - 0.271
2-aminobenzoyl-Ile-Ala-Ala-Gly-5-amino-2-nitrobenzoylamide
0.014 - 0.612
2-aminobenzoyl-Ile-Ala-Lys-Asp-5-amino-2-nitrobenzoylamide
0.0056 - 0.385
2-aminobenzoyl-Phe-Gly-Ala-Lys-5-amino-2-nitrobenzoylamide
0.033
cystatin C
Gly11 bond hydrolyzed by staphopain A, N-terminal truncation shown to impair inhibition of additional targets, disturbance of the host protease-inhibitor balance
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0.032
cystatin D
Ala10 bond hydrolyzed by staphopain A, truncation of cystatin D shown to cause alleviated inhibition of endogenous target enzymes investigated, disturbance of the host protease-inhibitor balance
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0.5
N-benzyloxycarbonyl-Phe-Leu-Glu-NH-p-nitroanilide
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pH 8.0-8.8
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3.3
N-Suc-Gly-Phe-Gly-p-nitroanilide
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used as substrate for staphopain inhibition studies
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.002 - 0.886
2-aminobenzoyl-Ile-Ala-Ala-Gly-5-amino-2-nitrobenzoylamide
0.012 - 0.089
2-aminobenzoyl-Ile-Ala-Lys-Asp-5-amino-2-nitrobenzoylamide
0.0112 - 0.716
2-aminobenzoyl-Phe-Gly-Ala-Lys-5-amino-2-nitrobenzoylamide
0.16
N-benzyloxycarbonyl-Phe-Leu-Glu-NH-p-nitroanilide
Staphylococcus aureus
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pH 8.0-8.8
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0.2
N-Suc-Gly-Phe-Gly-p-nitroanilide
Staphylococcus aureus
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used for inhibition studies of staphopain identified from a novel staphylococcal thiol protease operon stpAB2CH-91
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.1 - 118
2-aminobenzoyl-Ile-Ala-Ala-Gly-5-amino-2-nitrobenzoylamide
62.4
2-aminobenzoyl-Ile-Ala-Lys-Asp-5-amino-2-nitrobenzoylamide
Staphylococcus aureus
-
pH 7.6, 37C, staphopain C
12195
0.1 - 127.8
2-aminobenzoyl-Phe-Gly-Ala-Lys-5-amino-2-nitrobenzoylamide
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00052
E-64
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pH 8.0-8.8
additional information
additional information
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5
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substrate insoluble elastin
7.5
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substrate benzyl-Tyr-OEt
8 - 8.8
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substrate casein or hemoglobin
additional information
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pI: 9.4
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
13000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
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the enzyme staphopain B is synthesized as preproenzyme, the prefragment is cleaved resulting in the proenzyme, after secretion the proenzyme is proteolytically activated to the mature enzyme
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure determination of staphopain A in complex with inhibitor E64, folding pattern
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inactive mutant staphopain B in complex with inhibitor staphostatin B, 21C, vapour diffusion method with sitting drops, reservoir solution: 2 M ammonium sulfate, 5% isopropanol, 0.8% v/v 1 M guanidinium hydrochloride, 1-2 weeks, cryoprotection in buffer containing a 9:1 mixture of 2.8 M ammonium sulfate and (2R,3R)-(-)-2,3-butanediol, injected into the drops, X-ray structure determination
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purified recombinant mature staphopain B, sitting drop vapour diffusion method at 4C or 21C, equal volume of protein solution containing 15 mg/ml protein in 5mM Tris, pH 7.5, and reservoir solution containing 50 mM Bis-Tris, pH 6.5, 20% w/v PEG 4000, or 100 mM HEPES, pH 7.5, 21% w/v PEG 4000, and 10% v/v 2-propanol, are mixed, a few weeks, X-ray diffraction structure determination and analysis at 2.5-2.8 A resolution
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staphopain A
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; ion-exchange chromatography, gelfiltration, immobilized metal ion affinity chromatography for His-tagged protein
cysteine protease StpA2aur CH-91 purified by gel filtration
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from culture supernatant, apparant homogenity
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His-Select nickel affinity column chromatography
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native from culture supernatant
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native from culture supernatant; recombinant inactive mutant form, His-tagged, from Escherichia coli; recombinant proenzyme form as GST-fusion protein from Escherichia coli, affinity chromatography, cleavage by V8 proteases to mature enzyme form
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purified by gel filtration
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recombinant and native protein, gel filtration
recombinant co-expressed His-tagged staphopain A and inhibitor staphostatin A from Escherichia coli strain BL21(DE3) inclusion bodies by refolding and nickel affinity chromatography, method optimization
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recombinant GST-fusion prostaphopain B from Escherichia coli strain BL21(DE3) by glutathione affinity chromatography, gel filtration, and cleavage of the GST-tag followed by ultrafiltration
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recombinant protein, purified by gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
co-expression of His-tagged staphopain A and its inhibitor staphostatin A in Escherichia coli strain BL21(DE3) in inclusion bodies, recombinant enzyme is toxic for Escherichia coli cells, therefore a co-expression with the enzyme inhibitor is required for recombinant enzyme production, method optimization
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cysteine protease staphopain B of Staphylococcus warneri transformed into Escherichia coli strain BL21(DE3)pLysS, expression failed, mutant C21A used for recombinant protein production, expression in Escherichia coli strain BL21(DE3)
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DNA and partial amino acid sequence determination and analysis
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expressed in Escherichia coli
expressed in Escherichia coli strain ER2566
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expressed in Escherichia coli; expressed in Escherichia coli
expressed in Escherichia coli; overexpressed in Staphylococcus aureus, His-tagged version expressed in Escherichia coli
expression of large amounts of active wild-type enzyme in Escherichia coli is not possible, probably due to toxicity for the host; expression of wild-type enzyme as fusion protein in Escherichia coli, hybrid construct with a N-terminal GST-moiety linked to the proenzyme via a thrombin cleavable linker; overexpression of the inactive mutant in Escherichia coli BL21(DE3) as His-tagged protein
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prostaphopain B, expression as GST-fusion protein in Escherichia coli strain BL21(DE3)
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ssp and scp operons, genomic organization of ssp and scp genes, overview
ssp and scp operons, genomic organization of ssp and scp genes, overview, overexpression of scp operon genes in Escherichia coli
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Staphylococcus aureus UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant. ScpA is higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. Analysis of time-dependent ScpA amount during strain cultivation.
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
staphopain levels are decreased under biofilm-forming conditions
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C237A
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inactive ScpA mutant
C243A
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inactive SspB mutant
C237A
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inactive ScpA mutant
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C243A
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inactive SspB mutant
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C21A
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purification of native staphopain B unsuccessful since strain SW035 produces only minimally detectable protease activity, active-site mutation generated by site-directed mutagenesis, catalytically inactive mutant StpBwar C21A used for interaction studies
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant co-expressed His-tagged staphopain A and inhibitor staphostatin A from Escherichia coli strain BL21(DE3) inclusion bodies by treatment with 6 M guanidinium hydrochloride, and dilution in buffer, at pH 6.0, with 40% glycerol content or with arginine and Tween 20 supplementation, method optimization
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using 50 mM sodium phosphate, 150 mM sodium chloride, 1 mM EDTA, 0.5 M arginine (pH 6.0), 30% (v/v) glycerol, 1 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
pharmacology