Information on EC 3.4.21.64 - peptidase K

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
3.4.21.64
-
RECOMMENDED NAME
GeneOntology No.
peptidase K
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
Hydrolysis of keratin, and of other proteins with subtilisin-like specificity. Hydrolyses peptide amides
show the reaction diagram
hydrolysis of keratin, and of other proteins with subtilisin-like specificity
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY hide
39450-01-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
commercial preparation from Roche Diagnostics
-
-
Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
Tritirachium album Limber
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
-
the central event in prion infection is the conformational conversion of host-encoded cellular prion protein into the pathogenic isoform. For the in vitro replication of pathogenic prion protein by protein-misfolding cyclic amplification, cofactors are required that are not produced by lower organism, e.g. insects, but by mammalia, e.g. C57BL/6J mice. Brain-derived factors are also necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant prion protein. Cofactor activity of insect cell lysates becomes functional after proteinase K digestion and heat treatment, following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-prion protein replication by protein-misfolding cyclic amplification. Not only RNA, but also DNA, are the key components of protein-misfolding cyclic amplification, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids in insect cells
physiological function
-
proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms
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
AAPA + H2O
?
show the reaction diagram
-
molecular dynamics simulations of the proteinase K alone and in complex with the peptide substrate AAPA are performed to investigate the effect of substrate binding on the dynamics/molecular motions of proteinase K
-
-
?
Acetyl-Tyr ethyl ester + H2O
?
show the reaction diagram
-
-
-
-
-
Aldolase + H2O
Hydrolyzed aldolase
show the reaction diagram
-
-
-
-
-
Alkynyl carboxylates + H2O
?
show the reaction diagram
-
-
-
-
-
asialofetuin + H2O
?
show the reaction diagram
-
-
-
-
?
Bap protein + H2O
?
show the reaction diagram
-
degradation
-
-
?
Bovine ribonuclease + H2O
Hydrolyzed bovine ribonuclease
show the reaction diagram
-
-
-
-
-
Bovine serum albumin + H2O
?
show the reaction diagram
-
-
-
-
?
casein + H2O
?
show the reaction diagram
-
-
-
-
?
casein + H2O
hydrolyzed casein
show the reaction diagram
-
-
-
-
-
creatine kinase + H2O
?
show the reaction diagram
Tritirachium album Limber
-
inactivation of rabbit muscle creatine kinase by processing
-
?
fetuin + H2O
?
show the reaction diagram
-
-
-
-
?
Glucose dehydrogenase + H2O
Hydrolyzed glucose dehydrogenase
show the reaction diagram
-
upon proteolysis the enzyme is inactivated and the polypeptide chain is cleaved into 2 distinct fragments (K-protein, MW 26000 and K-peptide, MW 3000), the cleavage occurs in the C-terminal region of the polypeptide chain.-Leu-Ala-+-Ser-Ser-Glu is proposed as the cleavage site, the term -+- depicts the point of cleavage
upon proteolysis the enzyme is inactivated and the polypeptide chain is cleaved into 2 distinct fragments (K-protein, MW 26000 and K-peptide, MW 3000), the cleavage occurs in the C-terminal region of the polypeptide chain. Leu-Ala-+-Ser-Ser-Glu is proposed as the cleavage site, the term -+- depicts the point of cleavage
-
human growth hormone + H2O
?
show the reaction diagram
-
proteolytic activity and specificity of PK is maintained after its immobilization to magnetic particles
-
-
?
human sensitive prion protein Sc + H2O
?
show the reaction diagram
Keratin + H2O
?
show the reaction diagram
-
-
-
-
?
Keratin + H2O
Hydrolyzed keratin
show the reaction diagram
-
-
-
-
-
Lactate dehydrogenase + H2O
Hydrolyzed lactate dehydrogenase
show the reaction diagram
-
-
-
-
-
N-Acetylated amino acid esters + H2O
?
show the reaction diagram
-
-
-
-
-
N-Acetylated peptide esters + H2O
?
show the reaction diagram
-
-
-
-
-
N-succinyl-Ala-Ala-Ala-p-nitroanilide + H2O
N-succinyl-Ala-Ala-Ala + p-nitroaniline
show the reaction diagram
Tritirachium album Limber
-
-
-
?
N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide + H2O
N-succinyl-Ala-Ala-Pro-Leu + p-nitroaniline
show the reaction diagram
-
-
-
-
?
normal cellular prion protein + H2O
pathogenic cellular prion protein + ?
show the reaction diagram
-
in mouse brain
-
-
?
Oxidized insulin B-chain + H2O
Hydrolyzed oxidized insulin B-chain
show the reaction diagram
-
main cleavage sites: Gln4-His5, Ser9-His10, Leu11-Val12, Leu15-Tyr16, Leu17-Val18, Phe24-Phe25, Tyr26-Thr27
main cleavage sites: Gln4-His5, Ser9-His10, Leu11-Val12, Leu15-Tyr16, Leu17-Val18, Phe24-Phe25, Tyr26-Thr27
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p-nitrophenyl acetate + H2O
p-nitrophenol + acetate
show the reaction diagram
Tritirachium album Limber
-
-
-
-
?
poly(L-lactide) + H2O
?
show the reaction diagram
-
enzyme moves on the surface of substrate film to hydrolyze the film around it
-
-
?
prion protein + H2O
?
show the reaction diagram
pro-recombinant transglutaminase + H2O
?
show the reaction diagram
-
successful cleavage at the pro-sequence
-
-
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Propynyl benzoate + H2O
?
show the reaction diagram
-
-
-
-
-
ribonuclease A + H2O
proteolytic fragments
show the reaction diagram
Tritirachium album Limber
-
-
-
?
Serum albumin + H2O
Hydrolyzed serum albumin
show the reaction diagram
-
-
-
-
-
succinyl-AAPF-4-nitroanilide + H2O
?
show the reaction diagram
Succinyl-Ala-Ala-Ala 2-nitroanilide + H2O
?
show the reaction diagram
-
-
-
-
-
Succinyl-Ala-Ala-Ala 4-nitroanilide + H2O
?
show the reaction diagram
-
-
-
-
-
succinyl-Ala-Ala-Ala-p-nitroanilide + H2O
succinyl-Ala-Ala-Ala + p-nitroaniline
show the reaction diagram
-
-
-
-
?
Synthetic peptide substrates + H2O
?
show the reaction diagram
-
primarily specific against aromatic or hydrophobic amino acid residues at the carboxyl side of the splitting point, activity is markedly promoted by elongating the peptide chain to the N-terminal from the splitting point
-
-
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Urea-denatured hemoglobin + H2O
Hydrolyzed urea-denatured hemoglobin
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
Bap protein + H2O
?
show the reaction diagram
-
degradation
-
-
?
human sensitive prion protein Sc + H2O
?
show the reaction diagram
-
degradation, pathogenic isoform, sensitive prion protein complexes show higher molecular weight than resistant prions
-
-
?
Keratin + H2O
?
show the reaction diagram
-
-
-
-
?
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Calcium
SDS
-
stimulates hydrolysis of serum albumin in a dose-dependent manner, caused primarily by denaturation of the protein substrate, inactivates with an oligopeptide as substrate
Urea
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stimulates hydrolysis of serum albumin in a dose-dependent manner, caused primarily by denaturation of the protein substrate
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Carbobenzoxy-Ala-Ala-chloromethyl ketone
-
-
Chloromethyl ketone derivatives
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e.g. carboxybenzoyl-Ala-Gly-PheCH2Cl, carboxybenzoyl-Ala-PheCH2Cl
Cu2+
Tritirachium album Limber
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at equilibrium two to three copper ions bind stoichiometrically to PK and destroy its activity. Initial reversible and weak binding phase and a slower, irreversible abolition of activity with a half-time of 6 min at saturating copper ion concentrations. PK digestion of cellular prion proteins and other proteins in brain homogenate is inhibited in a concentration-dependent manner at concentrations of more than 1 mM. Presence of calcium ions, up to 10 mM, has no effect on copper inhibition
fructose 1,6-diphosphate
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mixed-type inhibition, more than one inhibitor molecule binds to proteinase K
glucose 6-phosphate
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mixed-type inhibition, more than one inhibitor molecule binds to proteinase K
Inhibitor from Helix aspersa
-
weak
-
Low-molecular-weight protein proteinase inhibitors from the granule-rich fraction of equine neutrophilic granulocytes
-
-
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MeOSuc-Ala-Ala-Ala-Pro-Phe-CH2Cl
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inhibits proteinase K (100 microgram/ml) at concentrations as low as 0.25 mM
MeOSuc-Ala-Ala-Pro-Phe-CH2Cl
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examination of inhibitory activity using a real-time reverse transcription-polymerase chain reaction assay in the presence of proteinase K. The AAPF inhibitor at a concentration of 0.05 mM allows a signal to be obtained for exogenous target Xeno RNA at 30 cycles
MeOSuc-Ala-Ala-Pro-Val-CH2Cl
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strong inhibitor
MeOSuc-Ala-Pro-Ala-Leu-CH2Cl
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weak inhibitor
N-Acetyl-L-Pro-L-Ala-L-Pro-L-Phe-D-Ala-L-Ala-NH2
-
substrate analogue
phenylmethylsulfonyl fluoride
-
-
Polyvalent proteinase inhibitor from albumin gland of Helix pomatia
-
-
-
Proteinase-inhibitors from the albumin gland of Achatina fulica
-
-
-
SDS
-
stimulates hydrolysis of serum albumin in a dose-dependent manner, caused primarily by denaturation of the protein substrate, inactivates with an oligopeptide as substrate
Turkey ovomucoid
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enzyme interacts with the third domain at the Leu18-Glu19 peptide bond, the reactive site of the inhibitor
-
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetonitrile
Tritirachium album Limber
-
-
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1
acetyl-(Ala)2-Ala methyl ester
-
-
0.8
acetyl-(Ala)2-Phe methyl ester
-
-
45
Acetyl-Ala methyl ester
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-
30
acetyl-Leu methyl ester
-
-
12
acetyl-Phe ethyl ester
-
-
5.6
acetyl-Trp ethyl ester
-
-
8.8
Acetyl-Tyr ethyl ester
-
-
26
acetyl-Val methyl ester
-
-
3.3
benzoyl-Arg ethyl ester
-
-
0.9
carboxybenzoyl-(Ala)2-Lys methyl ester
-
-
3
carboxybenzoyl-Ala-Lys methyl ester
-
carboxybenzoyl-Phe-Lys methyl ester
20
carboxybenzoyl-D-Ala-Lys methyl ester
-
-
19
carboxybenzoyl-Gly-Lys methyl ester
-
-
9
carboxybenzoyl-Leu-Lys methyl ester
-
-
21
carboxybenzoyl-Lys methyl ester
-
-
0.46 - 2.76
succinyl-AAPF-4-nitroanilide
1.29 - 4.23
succinyl-Ala-Ala-Ala-p-nitroanilide
additional information
additional information
-
-
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
51 - 827
succinyl-AAPF-4-nitroanilide
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5 - 12
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urea-denatured hemoglobin
7.5
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assay at
8 - 8.5
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assay at
10.5
-
-
additional information
-
pI: 8.9
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3 - 7
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proteinase K maintains structural integrity in the range of pH 3.0-7.0
6.7 - 7.4
Tritirachium album Limber
-
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8 - 11
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 50
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soluble and immobilized enzyme
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
secretion starts when the stationary phase of growth is reached, and when the culture medium is depleted of glucose and amino acids
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28930
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Tritirachium album, amino acid sequence analysis, the enzyme contains 2 disulfide bonds and a free cysteine residue
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 65500, precursor-protein, calculated, x * 34000, mature protein, SDS-PAGE
monomer
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1 * 28930, Tritirachium album, amino acid sequence analysis
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
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sequence consists of preproregion, catalytic domain and two C-terminal domains. Catalytic domain with the two C-terminal domains is an active protein of 56 kDa that converts at 50C by autolytic cleavage to mature 34 kDa protein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
at 0.15 nm resolution
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at 1.5 A resolution
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at 3.3 A resolution
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computer modeling studies indicates the presence of a small binding pocket in the vicinity of the proteinase K active site, which may be filled by the aromatic phenylalanine moiety
-
crystallographic study of its complex with a dipeptide chloromethyl ketone inhibitor
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Langmuir-Blodgett nanotemplate method and hanging drop vapor diffusion method, using 400 mM Na/K-tartrate in 25 mM HEPES pH 7.0
mercury-inhibited protein in presence of synthetic peptides GGGWGQPH and HGGGW, derived from N-terminal domain of human prion protein. Segment GGG is strongly bound as a substrate at the substrate recognition site
-
structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2 A resolution, N-acetyl-L-Pro-L-Ala-L-Pro-L-Phe-D-Ala-L-Ala-NH2
-
the crystallization of proteinase K using PEG 8000 is performed at various degrees of solvent deuteration and X-ray crystallographic analysis at 1.1 A resolution confirms that deuteration has no effect on crystal quality or crystal structure in this case
-
three-dimensional structure at 1.48 A resolution
-
three-dimensional structure of the complex of proteinase K with its naturally occuring protein inhibitor, PKI3
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to 1.32 A resolution. final model contains two Ca2+ ions, a molecule of digalacturonic acid and a partially occupied HEPES molecule. The digalacturonic acid molecule has one sugar moiety disposed exactly on a crystallographic twofold axis and is bound through hydrogen-bonding networks involving residue S150 and water molecules
-
to 2.3 A resolution, space group P43212
-
X-ray crystal structure at 1.5 A resolution shows that it has 2 binding sites for Ca2+
-
-
Tritirachium album Limber
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space group P4(3)2(1)2, cell dimensions a = b = 67.3 A, c = 106.6 A
Tritirachium album Limber
-
space group P4(3)2(1)2, cell dimensions a = b = 68.3, c = 108.4
Tritirachium album Limber
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.5
-
the enzyme acquires partially unfolded conformation at pH 2.5 with lower activity
707588
4 - 12
-
-
668577
5.5 - 9.5
-
-
668577
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
30 min, about 90% residual activity even in presence of 1% sodium dodecylsulfate
46
-
15 min, 50% loss of activity of Ca2+-free enzyme
60
Tritirachium album Limber
-
retains 83% activity after 2 h
65
-
15 min, 50% loss of activity of Ca2+-saturated enzyme
66
-
thermal unfolding is cooperative at pH 7.0 with transition midpoint of 66C
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Depletion of Ca2+ increases the rate of autolysis after about 48 h, it reduces the thermal stability and enhances the deactivation by 8 M urea
-
proteinase K is losing the proteolytic activity as the enzyme is attaining beta conformation
-
Relatively resistant to SDS, 0.2% in 50 mM Tris/HCl, pH 7.4
-
Relatively stable towards heat and denaturing agents
-
Urea, 4 M, stable
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
isopropanol
-
at 40% (v/v) isopropanol nearly half and at 50% (v/v) isopropanol all the tertiary structure is lost, beyond 30% (v/v) isopropanol there is some perturbation in the secondary structure of the protein
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, stable for at least 12 months
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutants purified over a Ni-NTA column
-
tagged cellular Bac-prion protein fusion partially 37fold from SF21 insect cells using immobilized metal ion affinity chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene encoding proteinase K re-synthesized with an Escherichia coli codon bias and cloned into an arabinose-inducible Escherichia coli expression vector
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recombinant expression of tagged cellular Bac-prion protein fusion in Spodoptera frugiperda SF21 insect cells via baculovirus transfection system
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A199S
-
no effect on activity
A236V
-
lethal
E138A
-
lethal, increased stability of the serine protease subtilisin
G293A
-
strong positive effect on activity
I132V
-
positive effect on activity
I310K
-
no effect on activity, increased stability of the serine protease subtilisin
K208H
-
positive effect on activity
K332R
-
positive effect on activity
L180I
-
positive effect on activity
L299C
-
lethal, increased stability of the serine protease subtilisin
M145F
-
no effect on activity, increased stability of the serine protease subtilisin
N95C
-
lethal, increased stability of the serine protease subtilisin
P265S
-
no effect on activity, increased stability of the serine protease subtilisin
P355S
-
no effect on activity, increased stability of the serine protease subtilisin
P97S
-
lethal, increased stability of the serine protease subtilisin
R237N
-
no effect on activity
S107D
-
no effect on activity
S123A
-
positive effect on activity
S273T
-
positive effect on activity
S337N
-
positive effect on activity
V167I
-
no effect on activity
V267I
-
positive effect on activity
Y151A
-
strong positive effect on activity
Y194S
-
no effect on activity, random mutation obtained during synthesis of wild-type proteinase K
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
biotechnology
degradation
-
use of enzyme to degrade poly(L-lactide) film. Adsorption of enzyme to film is irreversible, enzyme moves on the surface of substrate to hydrolyze the film around it
diagnostics
-
prion disease diagnosis relies on the relative resistance of sensitive prion protein Sc, PtPSc, to the non-specific protease proteinase K in brain samples to discriminate between resistant and senstive prions, PrPC and PrPSc, in combination with immunological detection of the main enzyme-resistant part of PrPSc (PrP27-30)
medicine
molecular biology
-
proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine shows a unique self-assembly in aqueous solutions
pharmacology
-
In human variant CreutzfeldtJakob disease, up to 90% of total prion protein present in the brain resists degradation with thermolysin, whereas only ?15% of this material resists digestion by proteinase K. Detection of proteinase K-sensitive isoforms of disease-related prion protein using thermolysin should be useful for improving diagnostic sensitivity in human prion diseases
synthesis
-
chemoenzymatic synthesis of oligo(L-phenylalanine) by the enzyme as a green and clean chemical reaction compared to organic synthesis