Information on EC 3.4.24.27 - thermolysin

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

EC NUMBER
COMMENTARY
3.4.24.27
-
RECOMMENDED NAME
GeneOntology No.
thermolysin
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
preferential cleavage: -/-Leu > -/-Phe
show the reaction diagram
-
-
-
-
preferential cleavage: -/-Leu > -/-Phe
show the reaction diagram
classical molecular dynamics simulation., and ab initio quantum mechanics/molecular mechanics, QM/MM, investigation of the Glu143-assisted water addition mechanism in thermolysin peptide hydrolysis. The mechanism consists of three distinct steps: (i) a Zn-bound water molecule is deprotonated by Glu143 and attacks the carbonyl bond of the substrate; (ii) Glu143 transfers the proton to the amide nitrogen atom; (iii) the nitrogen atom is protonated and the peptide bond is irreversibly broken. Transition state stabilization for nucleophilic attack is achieved by formation of a weak coordination bond between the substrate carbonyl oxygen atom and the Zn ion and of three strong hydrogen bonds between the substrate and protonated His231 and two solvent molecules, overview
-
preferential cleavage: -/-Leu > -/-Phe
show the reaction diagram
substrate turnover is associated with hinge bending that leads to a closed conformation. Product release regenerates the open form, such that steady-state catalysis involves a continuous closing/opening cycle. Elements in the periphery of the two lobes are most mobile. These peripheral regions undergo quite pronounced structural changes during the catalytic cycle. In contrast, active site residues exhibit only a moderate degree of backbone flexibility, and the central zinc appears to be in a fairly rigid environment. The hydrogen/deuterium exchange behavior of catalytically active thermolysin is indistinguishable from that of the free enzyme
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
hydrolysis of peptide bond
-
-
-
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Bacillus thermoproteolyticus neutral proteinase
-
-
-
-
EC 3.4.24.4
-
-
formerly
-
EC 3.4.24.4
-
formerly
NprM
Geobacillus stearothermophilus MK232
-
-
-
protease type X
Bacillus thermoproteolyticus Rokko
-
-
-
proteinase type X
-
-
proteinase type X
Bacillus thermoproteolyticus Rokko
-
-
-
Proteinase, Bacillus thermoproteolyticus neutral
-
-
-
-
Thermoase
-
-
-
-
Thermoase Y10
-
-
-
-
thermolysin-like protease
-
-
thermolysin-like protease
-
-
thermolysin-like protease
Geobacillus stearothermophilus MK232
-
-
-
Thermostable neutral proteinase
-
-
-
-
TL
Bacillus thermoproteolyticus Rokko
-
-
-
TLN
-
-
-
-
TLP-ste
Geobacillus stearothermophilus MK232
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9073-78-3
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
Bacillus proteolyticus
-
-
-
Manually annotated by BRENDA team
commercial preparation
-
-
Manually annotated by BRENDA team
enzyme immobilized on Celite
-
-
Manually annotated by BRENDA team
expressed in Escherichia coli
-
-
Manually annotated by BRENDA team
immobilized enzyme
-
-
Manually annotated by BRENDA team
subspecies Bacillus thermoproteolyticus rokko
-
-
Manually annotated by BRENDA team
Bacillus thermoproteolyticus Rokko
Rokko
-
-
Manually annotated by BRENDA team
strain MK232, genes npr and nprT
-
-
Manually annotated by BRENDA team
Geobacillus stearothermophilus MK232
strain MK232, genes npr and nprT
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
additional information
-
autodegradation at position 154155
additional information
-
life cycle of the thermolysin-like protease from Bacillus stearothermophilus in light of the calcium-dependent stability and instability of the N-terminal domain.
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 + H2O
?
show the reaction diagram
-
fluorescent substrate
-
?
(7-methoxycoumarin-4-yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 + H2O
?
show the reaction diagram
-
i.e. MOCAc-PLGL(Dpa)AR, fluorescent substrate
-
-
?
(7-methoxycoumarin-4yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH2 + H2O
?
show the reaction diagram
-
-
-
-
?
(europium(III) complex of a modified terpyridine)-K1K1K1-GFSAK1K1K-black hole quencher 2 + H2O
?
show the reaction diagram
-
thermolysin cleaves the substrates at the glycine-phenylalanine bond
-
-
?
(europium(III) complex of a modified terpyridine)-K1K2K2GFSAK2K-black hole quencher 2 + H2O
?
show the reaction diagram
-
thermolysin cleaves the substrates at the glycine-phenylalanine bond
-
-
?
(europium(III) complex of a modified terpyridine)-K1K2K2GFSAK2K2K-black hole quencher 2 + H2O
?
show the reaction diagram
-
thermolysin cleaves the substrates at the glycine-phenylalanine bond
-
-
?
1-beta-D-arabinofuranosyl-N4-lauroylcytosine + H2O
?
show the reaction diagram
-
-
-
-
?
11S soy protein+ H2O
?
show the reaction diagram
-
-
-
-
?
2-hydroxy-N-(4-methyl-2-nitrophenyl)-3-nitrobenzamide + H2O
?
show the reaction diagram
-
-
-
-
?
2-N-(4-[4'-N',N'-(dimethylamino)phenylazo]-benzoyl-L-serinyl-L-phenylalanylamido)-N''-ethylaminonaphthalene-5-sulfonic acid + H2O
?
show the reaction diagram
-
-
-
-
?
3-(2-furylacryloyl)-glycyl-L-leucine amide + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
5-bromo-N-(4-bromophenyl)-2-hydroxy-3-nitro-benzamide + H2O
?
show the reaction diagram
-
-
-
-
?
7S soy protein + H2O
?
show the reaction diagram
-
-
-
-
?
Ac-Gly-Leu-Ala-methylamide + H2O
?
show the reaction diagram
-
model substrate, enzyme-substrate complex, docking structures, overview
-
-
ir
alpha-1-antichymotrypsin + H2O
?
show the reaction diagram
-
cleavage within the sequence LSA-LVE
-
-
?
alpha-1-antitrypsin + H2O
?
show the reaction diagram
-
cleavage within the sequence AMF-LEA
-
-
?
azocasein + H2O
?
show the reaction diagram
-
-
-
-
?
azocasein + H2O
?
show the reaction diagram
-
-
-
-
?
Azocoll + H2O
?
show the reaction diagram
-
-
-
-
?
benzyloxycarbonyl-(4-nitro)Phe-Leu-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
benzyloxycarbonyl-Asp + Phe-methylester
benzyloxycarbonyl-Asp-Phe methyl ester + H2O
show the reaction diagram
-
-
-
-
benzyloxycarbonyl-Asp + Phe-methylester
benzyloxycarbonyl-Asp-Phe methyl ester + H2O
show the reaction diagram
-
-
-
-
benzyloxycarbonyl-Gly-(4-nitro)Phe-Leu-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
benzyloxycarbonyl-Gly-Gly-(4-nitro)Phe-Leu-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
benzyloxycarbonyl-Gly-Gly-Phe-Leu-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
benzyloxycarbonyl-Gly-Phe-Leu-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
benzyloxycarbonyl-Phe-Leu-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
bovine alpha-lactalbumin + H2O
?
show the reaction diagram
-
reaction at 25C and 70C under nonreducing conditions. At 25C, substrate undergoes limited hydrolysis leading to peptides no longer degraded. At 70C, protein is first quickly cleaved, then unfolded, leading to the release of intermediate peptides that may be further degraded
-
-
?
bovine beta-lactoglobulin A + H2O
?
show the reaction diagram
-
analysis of 25 peptides released by enzyme at 37C, comparison with peptides relased at 25C, 60 and 80C. Test of peptides for angiotensin-converting enzyme inhibiting activity
-
-
?
Carbobenzoxy-Gly-Pro-Leu-Ala-Pro + H2O
?
show the reaction diagram
-
-
-
-
-
casein + H2O
?
show the reaction diagram
-
from bovine milk
-
-
?
cellular prion protein + H2O
?
show the reaction diagram
-
thermolysin degrades cellular prion protein while preserving both proteinase K-sensitive and proteinase K-resistant isoforms of disease-related prion protein in both rodent and human prion strains. In variant Creutzfeldt-Jakob disease, up to 90% of total prion protein present in the brain resists degradation with thermolysin, whereas only about 15% of this material resists digestion by proteinase K
-
-
?
Dansyl peptides + H2O
?
show the reaction diagram
-
-
-
-
-
Dansyl-Gly-Phe-Ala + H2O
?
show the reaction diagram
-
-
-
-
-
DL-phenylalanine methyl ester + L-aspartic acid
D-phenylalanine methyl ester + L-alpha-aspartame
show the reaction diagram
Bacillus proteolyticus
-
-
-
-
-
F-Asp-PheOMe + H2O
?
show the reaction diagram
-
dipeptide synthesis
-
r
FA-glycyl-L-leucine amide + H2O
?
show the reaction diagram
-
-
-
?
furylacryloyl-Gly-Leu-NH2 + H2O
furylacryloyl-Gly + Leu-NH2
show the reaction diagram
-
-
-
-
-
GFA + H2O
Phe-Ala + Gly
show the reaction diagram
-
-
-
?
GFS + H2O
Gly + Phe-Ser
show the reaction diagram
-
-
-
?
GFS + H2O
Gly + Phe-Ser
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
GFSA + H2O
GF + FSA + ?
show the reaction diagram
-
-
-
?
GFSA + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
GFSAK + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
GFSAKN + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
GFSAKNQS + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
Gly-Phe-Leu + H2O
Phe-Leu + Gly-Phe + Gly + Leu
show the reaction diagram
-
-
-
?
H-Gly-Phe-Ala-OH + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
H-Gly-Phe-Leu-OH + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
H-Gly-Phe-Ser-Ala-Lys-Asn-Gln-Ser-Asn-Gln-Arg-OH + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
H-Gly-Phe-Ser-Ala-Lys-Asn-Gln-Ser-OH + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
H-Gly-Phe-Ser-OH + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
H-Gly-Ser-Ala-OH + H2O
?
show the reaction diagram
-
-
-
?
hide powder azure + H2O
?
show the reaction diagram
-
-
-
-
?
leucine enkephalin + H2O
?
show the reaction diagram
-
-
-
-
-
leucine enkephalin + H2O
?
show the reaction diagram
-
-
-
?
leucine enkephalin + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus Rokko
-
-
-
-
-
N'-[3-(2-furyl)acryloyl]glycyl-L-leucinamide + H2O
?
show the reaction diagram
-
-
-
-
?
N-(2,3-dimethylphenyl)-2-hydroxy-3-nitro-benzamide + H2O
?
show the reaction diagram
-
-
-
-
?
N-(2,4-dimethylphenyl)-2-hydroxy-3-nitro-benzamide + H2O
?
show the reaction diagram
-
-
-
-
?
N-(2,5-dimethylphenyl)-2-hydroxy-3-nitrobenzamide + H2O
?
show the reaction diagram
-
-
-
-
?
N-(2-chloro-4-nitrophenyl)-2-hydroxy-3-nitro-benzamide + H2O
?
show the reaction diagram
-
-
-
-
?
N-(2-chloro-6-methylphenyl)-2-hydroxy-3-nitrobenzamide + H2O
?
show the reaction diagram
-
-
-
-
?
N-(2-ethylphenyl)-2-hydroxy-3-nitrobenzamide + H2O
?
show the reaction diagram
-
-
-
-
?
N-(4-methoxyphenylazoformyl)-Leu-Leu-OH + H2O
?
show the reaction diagram
Geobacillus stearothermophilus, Geobacillus stearothermophilus MK232
-
a synthetic substrate
-
-
?
N-(5-chloro-2-methoxyphenyl)-2-hydroxy-3-nitro-benzamide + H2O
?
show the reaction diagram
-
-
-
-
?
N-(Benzyloxycarbonyl)-L-Phe + L-Phe methyl ester
N-(Benzyloxycarbonyl)-L-Phe-L-Phe methyl ester + H2O
show the reaction diagram
-
-
-
-
N-(Benzyloxycarbonyl)-L-Phe + L-Phe methyl ester
N-(Benzyloxycarbonyl)-L-Phe-L-Phe methyl ester + H2O
show the reaction diagram
-
-
-
-
N-benzyloxycarbonyl-Gly-L-Leu amide + H2O
N-benzyloxycarbonyl-Gly + L-Leu amide
show the reaction diagram
-
-
-
-
?
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester + H2O
N-benzyloxycarbonyl-L-Asp + L-Phe-methyl ester
show the reaction diagram
-
-
-
-
?
N-carbobenzoxy-Gly-L-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
?
N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
N-carbobenzoxy-L-Asp-L-Phe methyl ester + H2O
?
show the reaction diagram
Geobacillus stearothermophilus MK232
-
-
-
-
?
N-carbobenzoxy-L-Asp-L-Phe-methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
N-carbobenzoxy-L-Asp-L-Phe-methyl ester + H2O
N-carbobenzoxy-L-Asp + L-Phe-methyl ester
show the reaction diagram
-
-
-
-
?
N-carbobenzoxy-L-Asp-L-Phe-methyl ester + H2O
N-carbobenzoxy-L-aspartic acid + L-phenylalanine methyl ester
show the reaction diagram
-
-
-
-
r
N-carbobenzyloxy-L-Asp-L-Phe methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
N-carboxybenzoyl-L-aspartyl-L-phenylalanine methyl ester + H2O
?
show the reaction diagram
-
-
-
?
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide + H2O
?
show the reaction diagram
-
-
-
-
?
N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
?
N-[3-(2-furyl)acryloyl]-Gly-L-Leu-NH2 + H2O
?
show the reaction diagram
-
-
-
-
?
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide + H2O
?
show the reaction diagram
-
-
-
r
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide + H2O
?
show the reaction diagram
-
i.e. FAGLA
-
-
?
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide + H2O
N-[3-(2-furyl)acryloyl]-glycine + L-leucine amide
show the reaction diagram
-
-
-
-
?
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide + H2O
N-[3-(2-furyl)acryloyl]-glycine + L-leucine amide
show the reaction diagram
-
-
-
-
?
N-[3-(2-furyl)acryloyl]-L-leucine-L-alanine amide + H2O
?
show the reaction diagram
-
i.e. FALAA
-
-
?
N-[3-(2-furyl)acryloyl]-Phe-Ala amide + H2O
?
show the reaction diagram
-
-
-
?
N-[3-(2-furyl)acryloyl]glycyl-L-leucinamide + H2O
?
show the reaction diagram
-
-
-
-
?
Nalpha-benzyloxycarbonyl-L-aspartyl-L-phenylalanine methyl ester + H2O
?
show the reaction diagram
-
-
-
?
Oxidized insulin B-chain + H2O
?
show the reaction diagram
-
major cleavage at the peptide bonds of His5-Leu6, His10-Leu11, Ala14-Leu15, Tyr16-Leu17, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, Phe25-Tyr26
-
-
-
oxyquinoline + H2O
?
show the reaction diagram
-
-
-
-
?
Pro-urokinase + H2O
?
show the reaction diagram
-
thermolysin activates thrombin-inactivated pro-urokinase nearly as rapidly as it does the native zymogen, cleavage of Arg156-Phe157 and Lys158-Ile159
-
-
-
soy protein isolate + H2O
?
show the reaction diagram
-
-
-
-
?
tryptic hydrolysate of bovine beta casein + H2O
?
show the reaction diagram
-
-
-
-
?
Tyr-Gly-Gly-Phe-Leu + H2O
?
show the reaction diagram
-
-
-
?
Z-Arg-PheOMe + H2O
?
show the reaction diagram
-
dipeptide synthesis
-
?
[3H]leucine enkephalin + H2O
?
show the reaction diagram
Bacillus thermoproteolyticus, Bacillus thermoproteolyticus Rokko
-
-
-
?
Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)-OH + H2O
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
-
-
-
-
additional information
?
-
-
specificity overview: various synthetic peptides
-
-
-
additional information
?
-
-
specificity overview: oligopeptides
-
-
-
additional information
?
-
-
specificity overview: hydrolyzes peptide bonds with amino groups of hydrophobic amino acids (Phe, Leu, Ala, Val, Ile but not Trp), specificity overview: no exopeptidase activity
-
-
-
additional information
?
-
-
enzyme additionally catalyzes the transesterification of vinyl laurate to several sucrose-containing tri- and tetrasaccharides. Preferred position of acylation is the 2-OH group of the alpha-D-glucopyranose moiety linked 1 to 2 to the beta-D-fructofuranose unit
-
-
-
additional information
?
-
-
enzyme catalyzes the formation of beta-cyclodextrin esters using vinyl esters of butyrate, decanoate and laurate, as acyl donors in dimethylsulfoxide. Esterification occurs exclusively at the glucose C-2 position. Enzyme also catalyzes the synthesis of alpha-, beta-, gamma- and maltosyl-beta-cyclodextrin esters with vinyl laurate as the acyl donor in dimethylsulfoxide and dimethylformamide
-
-
-
additional information
?
-
-
enzyme prefers basic resiudes in P3 position
-
-
-
additional information
?
-
-
thermolysin selectively digests the dermo-epidermal junction of epidermal sheets used for burnt persons, overview
-
-
-
additional information
?
-
-
digestion of brain homogenates from healthy and scrapie-affected sheep as well as healthy and BSE-affected cattle, the PK digestion results in the amino-terminal truncation of PrPSc-producing PrP species with molecular weights of approx 17, 21, and 27 kDa that can only be detected with antibodies binding to the carboxy-terminal region of PrP, the so-called protease-resistant core or PrP27-30, overview
-
-
-
additional information
?
-
-
thermolysin is a thermostable neutral metalloproteinase and performs autocatalytic cleavage for pro-enzyme activation
-
-
-
additional information
?
-
-
thermolysin performs Co2+-stimulable autolysis
-
-
-
additional information
?
-
-
preferentially, peptide bonds preceding hydrophobic residues are hydrolyzed, such as I, L or F at P1' position. The enzyme less frequently also cleaves substrates with hydrophilic residues such as E, T or Q in the P1' position
-
-
-
additional information
?
-
-
the major site for thermolysin cleavage specificity, (the S1' site), accepts large hydrophobic residues. Thermolysin preferentially cleaves at the N-terminal side of hydrophobic or bulky amino side chains such as Leu, Phe, Ile and Val. Thermolysin also cleaves bonds of Met, His, Tyr, Ala, Asn, Ser, Thr, Gly, Lys, Glu or Asp at the P1' site
-
-
-
additional information
?
-
-
autodegradation at position 154155
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
N-carbobenzoxy-L-Asp-L-Phe-methyl ester + H2O
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
thermolysin selectively digests the dermo-epidermal junction of epidermal sheets used for burnt persons, overview
-
-
-
additional information
?
-
-
autodegradation at position 154155
-
-
-
METALS and IONS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
Ca2+
-
there are two adjacent calcium ions seemingly firmly bound inside the surface of the molecule by chelation to five acidic groups: Asp138, Glu177, Asp185, Glu190 and Asp191, two additional calcium binding sites are at exposed surface regions, one chelated by Asp57 and possibly also by Asp59 and the other chelated by Asp200
Ca2+
-
contains 4 structural calcium ions
Ca2+
-
4 calcium ions required for structural stability
Ca2+
-
required for stability
Ca2+
-
four Ca2+ per enzyme molecule are required for enzyme stability
Ca2+
-
four ions per enzyme molecule required for stability
Ca2+
-
four Ca2+ ions are required for thermostability
Ca2+
-
thermolysin has four calcium ions responsible for its thermostability
Ca2+
-
thermolysin consists of four Ca2+ ligand ions necessary for stability
Ca2+
-
TLN contains one zinc ion and four calcium ions, and these ions contribute to enzymatic catalysis and structural stability
Ca2+
-
required
Ca2+
-
protease binds calcium ions in the regions that are involved in the autolytic maturation process, at least one of the calcium ions plays a regulatory role. This calcium ion plays an important role as a switch that modulates the protease between stable and unstable states as appropriate to the biological need
Co2+
-
2fold activation at 0.4 mM
Co2+
-
active zinc ion in thermolysin can be substituted by Co2+, doubles activity
Co2+
-
increases the activity 3-4fold at up to 2 mM, but inhibits at 2-18 mM, activation-and-inhibition dual effects of Co2+ ion are analysed kinetically, Co2+-dependent activation is inhibited competitively by Zn2+ ion at 0.0001-0.001 mM
K+
-
activates, preference of monovalent cations in descending order: Na+, K+, Li+
Na+
-
activates, preference of monovalent cations in descending order: Na+, K+, Li+, the bell-shaped pH dependence profile of the FAGLA-hydrolyzing activity of thermolysin is shifted from pH 5.4 to pH 6.7 by the addition of 4 M NaCl
Na+
-
4 M Na+ stimulates the hydrolytic activity of wild type thermolysin about 13fold
NaBr
-
4 M enhances activity 13-15 times
NaCl
-
activity is remarkably enhanced by 1-5 M neutral salts
NaCl
-
4 M enhances activity 13-15 times
NaCl
-
induces activation
NaCl
-
activates the mutant enzymes at 4 M to 17-19fold of wild-type enzyme activity, overview
NaCl
-
induces enzyme activation, activation of mutant enzymes is reduced compared to the wild-type enzyme
NaSCN
-
3fold increase in catalytic activity of thermolysin when the NaSCN concentration is increased to 1 M, but decrease in catalytic activity at higher concentrations of NaSCN
Zinc
-
zinc metalloproteinase
Zinc
-
the three zinc ligands are two histidines and glutamic acid
Zn2+
-
zinc-metallopeptidase
Zn2+
-
Zn-dependent
Zn2+
-
zinc-containing neutral metalloendoprotease
Zn2+
-
active zinc ion in thermolysin
Zn2+
-
required for enzyme activity
Zn2+
-
zinc endopeptidase
Zn2+
-
required for activity
Zn2+
-
a zinc metalloproteinase that contains a HEXXH motif, one Zn2+ per enzyme molecule is required for activity
Zn2+
-
a zinc metalloproteinase
Zn2+
-
stereochemical relationships between Gln128, Glu143, Gln225, Asp226, His231 and active site Zn2+ of thermolysin, overview
Zn2+
-
one ion per enzyme molecule required for activity
Zn2+
-
one ion per enzyme molecule essential for activity, the enzyme contains the HEXXH motif constituting the zinc catalytic site
Zn2+
-
a zinc metalloprotease
Zn2+
-
zinc metalloproteinase, one ion per enzyme molecule required for activity
Zn2+
-
residues H142, H146, and E166 coordinate the catalytic zinc
Zn2+
-
comparison of metal preferences of Escherichia coli peptide deformylase and Bacillus thermoproteolyticus thermolysin. Both enzymes catalyze via the same chemical steps, and reproduce their different preferences for zinc or iron as competent cofactors. In thermolysin, the substrate is strongly activated and can serve as the fifth coordination ligand of zinc prior to the chemical steps. When iron replaces zinc, its stronger interaction with the hydroxide ligand may lead to higher activation barrier in thermolysin
Zn2+
-
dependent on
Zn2+
-
a single catalytic Zn2+ ion is essential for hydrolytic activity
Zn2+
-
thermolysin consists of three Zn2+ ligand ions necessary for activity
Zn2+
-
TLN contains one zinc ion and four calcium ions, and these ions contribute to enzymatic catalysis and structural stability
Zn2+
-
dependent on, thermolysin is a bacterial zinc metalloproteinase. The active site zinc atom is tetrahedrally coordinated when the inhibitors N-benzyloxycarbonyl-tryptophan or N-benzyloxycarbonyl-phenylalanine are bound to thermolysin
Li+
-
activates, preference of monovalent cations in descending order: Na+, K+, Li+
additional information
-
mechanism of salt-induced activation
additional information
-
thermolysin activity as well as its stability is remarkably enhanced by high concentration of neutral salts consisting of Na+, K+, Cl-, and Br- in the synthesis and hydrolysis of N-carbobenzoxy-L-aspertyl-L-phenylalanine methyl ester and hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1,10-phenanthroline
-
5 mM, complete inhibition
1-beta-D-arabinofuranosyl-N4-lauroylcytosine
-
competitive
1-butanol
-
-
1-Propanol
-
-
2-(4-chlorophenyl) quinazolin-4(3H)-one
-
low inhibitory activity
-
2-(4-methylphenyl) quinazolin-4(3H)-one
-
low inhibitory activity
-
2-(4-methylphenyl)-3-(1,3-thiazol-2-yl) quinazolin-4(3H)-one
-
-
-
2-(4-oxo-2-methylquinazolin-3(4H)-yl) guanidine
-
low inhibitory activity
-
2-(acetyloxy)-3-chlorobenzoic acid
-
-
-
2-(N-Bromoacetyl-N-hydroxyamino)-4-methylpentanonitrile
-
irreversible
2-aminobenzoyle-N-[3-(4-oxo-2-phenylquinazolin-3(4H)-yl) propyl]benzamide
-
low inhibitory activity
-
2-butanol
-
-
2-chloro-N-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetamide
-
low inhibitory activity
-
2-ethyl-3-hydroxyquinazolin-4(3H)-one
-
-
-
2-ethylquinazolin-4(3H)-one
-
-
-
2-hydroxy-N-(4-methyl-2-nitrophenyl)-3-nitrobenzamide
-
competitive
2-methyl-1-propanol
-
-
2-methyl-1-propanol
-
-
2-phenyl-3-(1, 3-thiazol-2-yl) quinazolin-4(3H)-one
-
low inhibitory activity
-
2-phenyl-3-[[(1E)-phenylmethylene] amino]-2,3-dihydroquinazolin-4(1H)-one
-
low inhibitory activity
-
2-phenylpropionyl-L-phenylalanine
-
-
-
2-phenylpropionyl-Leu-Trp
-
-
-
3-(2-hydroxyethyl) quinazolin-4(3H)-one
-
low inhibitory activity
-
3-(3-aminoethyl)-2-(4-methylphenyl) quinazolin-4(3H)-one
-
low inhibitory activity
-
3-(3-aminopropyl)-2-(4-methylphenyl) quinazolin-4(3H)-one
-
low inhibitory activity
-
3-(4-iodophenyl)-2-phenylquinazolin-4(3H)-one
-
low inhibitory activity
-
3-(isopropylideneamino)-2,2-dimethyl-2,3-dihydroquinazolin-4(1H)-one
-
potent inhibitor
-
3-([(1E)-[4-(dimethylamino) phenyl]methylene]amino)-2-methylquinazolin-4(3H)-one
-
-
-
3-([(1E)-[4-(dimethylamino) phenyl]methylene]amino)-2-phenylquinazolin-4(3H)-one
-
-
-
3-amino-2-(4-chlorophenyl)quinazolin-4(3H)-one
-
-
-
3-amino-2-(4-nitrophenyl)quinazolin-4(3H)-one
-
-
-
3-amino-2-(hydrazinomethyl) quinazolin-4(3H)-one
-
low inhibitory activity
-
3-amino-2-(trifluoromethyl) quinazolin-4(3H)-one
-
-
-
3-amino-2-methylquinazolin-4(3H)-one
-
-
-
3-hydroxy-2-isopropylquinazolin-4(3H)-one
-
-
-
3-hydroxy-2-methylquinazolin-4(3H)-one
-
-
-
3-methylaspirin
-
-
-
3-phenyl-2-(trifluoromethyl) quinazolin-4(3H)-one
-
potent inhibitor
-
3-Phenylpropionyl-L-Phe
-
crystallographic study of the binding to thermolysin
3-[[(1E)-(2-hydroxynaphthalen-1-yl)methylene]amino]-2-phenylquinazolin-4(3H)-one
-
low inhibitory activity
-
3-[[(1E)-(3-chlorophenyl)methylene]amino]-2-phenylquinazolin-4(3H)-one
-
-
-
3-[[(1E)-(4-fluorophenyl)methylene]amino]-2-phenylquinazolin-4(3H)-one
-
low inhibitory activity
-
4-methyl-N-(2-methyl-4-oxoquinazolin-3(4H)-yl)benzamide
-
low inhibitory activity
-
5-bromo-N-(4-bromophenyl)-2-hydroxy-3-nitro-benzamide
-
competitive
alpha2-Macroglobulin
-
-
-
Bifunctional N-carboxyalkyl dipeptides
-
-
-
Carbobenzoxy-L-Phe
-
crystallographic study of the binding to thermolysin
Cbz-Gly-PSI[P(O)OH]-Leu-Leu
-
-
Cbz-Phe-PSI[P(O)OH]-Leu-Ala
-
-
ClCH2CO-DL-(N-OH)Leu-OCH3
-
specific, irreversible, pH-dependence of inhibition
Co2+
-
increases the activity 3-4fold at up to 2 mM, but inhibits at 2-18 mM, activation-and-inhibition dual effects of Co2+ ion are analysed kinetically
Co2+
-
competitive, 72% inhibition at 18 mM, partial protection or reverse effect by Ca2+ at 0.5 mM, Co2+ show partly Ca2+-sensitive and partly Ca2+-insensitive inhibition, molecular mechanism, Co21 accelerates the autolysis, overview
Cu2+-Cys-Gly-His-Lys
-
stimulation at concentration up to 0.01 mM, inhibition at higher concentrations of 0.01-0.1 mM, binding and kinetics, overview
Dipeptides
-
overview, temperature dependence, pH-dependence
Dipeptides
-
crystallographic study of the binding to thermolysin
EDTA
-
5 mM, complete inhibition
ethanimidic acid N-[4-oxo-2-phenyl-3(4H)-quinazolinyl]-ethyl ester
-
-
-
ethyl (4-oxo-3,4-dihydroquinazolin-2-yl)acetate
-
low inhibitory activity
-
HONH-benzylmalonyl-L-Ala-Gly-NH2
-
-
-
Hydroxamic acid inhibitors
-
binding to thermolysin suggests a pentacoordinate zinc intermediate
-
N-(1-Carboxy-3-phenylpropyl)-Leu-Trp
-
-
N-(2,3-dimethylphenyl)-2-hydroxy-3-nitro-benzamide
-
competitive
N-(2,4-dimethylphenyl)-2-hydroxy-3-nitro-benzamide
-
competitive
N-(2,5-dimethylphenyl)-2-hydroxy-3-nitrobenzamide
-
competitive
N-(2-chloro-4-nitrophenyl)-2-hydroxy-3-nitro-benzamide
-
competitive
N-(2-chloro-6-methylphenyl)-2-hydroxy-3-nitrobenzamide
-
competitive
N-(2-ethylphenyl)-2-hydroxy-3-nitrobenzamide
-
competitive
N-(2-methyl-4-oxoquinazolin-3(4H)-yl)acetamide
-
low inhibitory activity
-
N-(2-methyl-4-oxoquinazolin-3(4H)-yl)benzamide
-
low inhibitory activity
-
N-(2-phenyl-4-oxoquinazolin-3(4H)-yl)acetamide
-
low inhibitory activity
-
N-(4-oxo-2-phenyl-3(4H)-quinazolininyl)-N-(3-nephthyl) acetamidine
-
low inhibitory activity
-
N-(5-chloro-2-methoxyphenyl)-2-hydroxy-3-nitro-benzamide
-
competitive
N-Benzyloxycarbonyl-L-phenylalanine
-
binding mechanism in aqueous solution, NMR and spectrophotometric analysis, overview. Substitution of Zn2+ by Co2+ decreases the binding affinity of the inhibitor, overview
N-benzyloxycarbonyl-L-tryptophan
-
binding mechanism in aqueous solution, NMR and spectrophotometric analysis, overview. Substitution of Zn2+ by Co2+ decreases the binding affinity of the inhibitor, overview. With thermolysin, a 1 M concentration of NaSCN produces an 2fold increase in its Ki value from 0.087 mM to 0.19 mM for the inhibitor N-benzyloxycarbonyl-tryptophan
-
N-chloroacetyl-N-hydroxyleucine methyl ester
-
irreversible inhibition
-
N-chloroacetyl-N-hydroxyleucyl-alanyl-glycinamide
-
irreversible inhibition
-
n-Pentanol
-
saturation concentration of activation at 60%, inhibition at higher concentration
N-Phosphoryl-Ile-Ala-OH
-
-
N-Phosphoryl-L-Leu amide
-
thermolysin-inhibitor complexes examined by NMR spectroscopy
N-Phosphoryl-L-Leu amide
-
-
N-Phosphoryl-L-Leu-L-Trp
-
specific inhibitor
N-Phosphoryl-Leu-Phe-OH
-
-
N-Phosphoryl-Leu-Trp-OH
-
-
N-[[[benzyloxycarbonyl]amino]methyl]hydroxyphosphinyl-L-Phe
-
-
Peptide hydrazides
-
-
-
Peptide hydroxamic acids
-
-
-
Peptides containing zinc coordination ligands
-
-
-
Phosphonamidates
-
-
-
Phosphonamidates
-
overview
-
phosphoramidon
-
i.e. N-(alpha-L-rhamnopyranosyloxyphospho)-L-Leu-L-Trp, thermolysin-inhibitor complexes examined by NMR spectroscopy
phosphoramidon
-
specific inhibitor; the OH-group is not essential for the binding to thermolysin
phosphoramidon
-
crystallographic study of the complex of phosphoramidon with thermolysin
phosphoramidon
-
strong competitive inhibitor
phosphoramidon
-
-
Zincov
-
competitive inhibitor
[(2S)-2-sulfanyl-3-phenylpropanoyl]Gly-(5-Ph)Pro
-
-
[(2S)-2-sulfanyl-3-phenylpropanoyl]Phe-Tyr
-
-
[(2S,R)-2-sulfanylheptanoyl]Phe-Ala
-
-
[Co(acacen)(NH3)2]Cl
-
irreversible inhibition
methyl 2-[(trifluoroacetyl) amino] benzoate
-
low inhibitory activity
-
additional information
-
not inhibitory: human blood serpins alpha-1-antitrypsin and alpha-1-antichymotrypsin
-
additional information
-
identification of inhibitors by structure based virtual screening and study of binding modes by docking
-
additional information
-
not inhibited by 2-phenyl-3-[(E)-phenyldiazenyl]-1,2,3,4-tetrahydro-10H[1,2,4]triazino[6,1b]-quinazolin-10-one, 3-mesityl-2-phenylquinazolin-4(3H)-one, 3-(2-hydroxyethyl)-2-methylquinazolin-4(3H)-one, and 3-amino-2-phenylquinazolin-4(3H)-one
-
additional information
-
not inhibited by 3-hydroxy-2-methylthieno[3,2-d]pyrimidine-4(3H)-one and 3-(acetylamino)-4-methylthiophene-2-carboxylic acid
-
additional information
-
the active site zinc atom is tetrahedrally coordinated when the inhibitors N-benzyloxycarbonyl-tryptophan or N-benzyloxycarbonyl-phenylalanine are bound to thermolysin. pH dependencies of inhibitors N-benzyloxycarbonyl-tryptophan or N-benzyloxycarbonyl-phenylalanine and binding to thermolysin, detailed overview
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
Ca2+
-
accelerated hydrolysis of some tryptic peptides derived from bovine beta-casein in presence of Ca2+, while other peptides are not affected
Calcium
-
supplementation of growth medium by calcium induces expression. Regulation by calcium ions is at posttranscriptional level
Cu2+-Cys-Gly-His-Lys
-
stimulation at concentration up to 0.01 mM, inhibition at higher concentrations of 0.01-0.1 mM, binding and kinetics, overview
n-Pentanol
-
saturation concentration of activation at 60%, inhibition at higher concentration
NaCl
-
up to 40fold increase in activity in presence of 4 M NaCl, substrate MOCAc-PLGL(Dpa)AR. Degree of activation depends on substrate
Cys-Gly-His-Lys
-
activation
additional information
-
direct effect of organic solvents on the microenvironment of the enzyme largely depends on the molecular structure of the solvent
-
additional information
-
no change in hydrolysis of tryptic peptides derived from beta-casein by presence of Na+
-
additional information
-
mechanism of salt-induced activation
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.002
-
(7-methoxycoumarin-4yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH2
-
pH and temperature not specified in the publication
0.00449
-
(europium(III) complex of a modified terpyridine)-K1K2K2GFSAK2K-black hole quencher 2
-
pH and temperature not specified in the publication
-
0.65
0.72
Benzyloxycarbonyl-(4-nitro)Phe-Leu-Ala
-
depending on mode of preparation of the form of enzyme
2.8
-
Benzyloxycarbonyl-Asp
-
pH 5.0 or pH 6.0
0.033
-
Benzyloxycarbonyl-Asp-Phe methyl ester
-
pH 5.5
0.059
-
Benzyloxycarbonyl-Asp-Phe methyl ester
-
pH 6.0
0.076
-
Benzyloxycarbonyl-Asp-Phe methyl ester
-
pH 5.0
0.98
1.39
Benzyloxycarbonyl-Gly-Phe-Leu-Ala
-
depending on mode of preparation of the form of enzyme
0.55
1.52
Benzyloxycarbonyl-Phe-Leu-Ala
-
depending on mode of preparation of the form of enzyme
0.2
-
dansyl-Ala-Ala-Phe-Ala
-
-
2
-
dansyl-Ala-Leu-Ala
-
-
0.91
-
dansyl-Ala-Phe-Ala
-
-
13
-
dansyl-Gly-Gly-Leu-Gly
-
-
5
-
dansyl-Gly-Leu-Gly
-
-
0.69
-
dansyl-Gly-Leu-Phe
-
-
0.08
0.09
Dansyl-Gly-Phe-Ala
-
depending on assay method
0.77
-
dansyl-Gly-Phe-Gly
-
-
0.3
-
dansyl-Gly-Phe-Phe
-
-
0.3
-
GFA
-
pH 8.0, 37C
0.6
-
GFA
-
pH 8.0, 37C, 5 M glycerol
0.4
-
GFL
-
pH 8.0, 37C, product FL
0.7
-
GFL
-
pH 8.0, 37C, 5 M glycerol, product GL
1.8
-
GFL
-
pH 8.0, 37C, 5 M glycerol, product FL
2.5
-
GFL
-
pH 8.0, 37C, 5 M glycerol, product GF
1.3
-
GFS
-
pH 8.0, 37C
3.5
-
GFS
-
pH 8.0, 37C, 5 M glycerol
0.7
-
GFSA
-
pH 8.0, 37C, product FSA
1.2
-
GFSA
-
pH 8.0, 37C, 5 M glycerol, product GF
6.8
-
GFSA
-
pH 8.0, 37C, 5 M glycerol, product FSA
12
-
GFSA
-
pH 8.0, 37C, 5 M glycerol, product GF
250
-
L-Phe methyl ester
-
pH 5.5 or 6.0
300
-
L-Phe methyl ester
-
-
5.8
-
L-phenylalanine methyl ester
-
mutant enzyme D150E, at pH 7.5, at 25C
7
-
L-phenylalanine methyl ester
-
mutant enzyme D150W, at pH 7.5, at 25C
7.3
-
L-phenylalanine methyl ester
-
mutant enzyme D150A, at pH 7.5, at 25C
8.7
-
L-phenylalanine methyl ester
-
wild type enzyme, at pH 7.5, at 25C
8.8
-
L-phenylalanine methyl ester
-
mutant enzyme N227H, at pH 7.5, at 25C
11.3
-
L-phenylalanine methyl ester
-
mutant enzyme F114A, at pH 7.5, at 25C
12.9
-
L-phenylalanine methyl ester
-
mutant enzyme I168A, at pH 7.5, at 25C
0.2
-
leucine enkephalin
-
pH 6.8, 37C, wild-type
0.21
-
leucine enkephalin
-
pH 7.4, 37C, wild-type
0.24
-
leucine enkephalin
-
pH 6.8, 37C, mutant D170A; pH 6.8, 37C, mutant R203M
0.48
-
leucine enkephalin
-
pH 7.4, 37C, mutant Y157F
20
-
N-(Benzyloxycarbonyl)-L-Phe
-
-
39.2
-
N-(Benzyloxycarbonyl)-L-Phe
-
-
12
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155S/I156N, pH 7.0, 37C; recombinant mutant L155S/I156V, pH 7.0, 37C
-
13
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155A/I156N, pH 7.0, 37C; recombinant mutant L155A/I156V, pH 7.0, 37C; recombinant mutant L155S, pH 7.0, 37C
-
14
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant wild-type enzyme, pH 7.0, 37C
-
20
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155A, pH 7.0, 37C
-
0.42
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant wild-type enzyme, pH 7.5, 25C
-
0.44
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117E, pH 7.5, 25C
-
0.5
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117R, pH 7.5, 25C
-
0.57
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117K, pH 7.5, 25C
-
10
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
mutant L155G, 37C, pH 7.0
13
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
mutant L155S, 37C, pH 7.0
14
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
wild-type, 37C, pH 7.0
17
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
mutant L155F, 37C, pH 7.0
20
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
mutant L155A, 37C, pH 7.0
0.06
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.0, 25C, recombinant mutant L144S
0.1
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.0, 25C, recombinant mutant G8C/N60C/S65P/L144S
0.16
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S/D150E/S53D, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.17
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S/D150E, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.18
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.0, 25C, recombinant mutant G8C/N60C/S65P
0.18
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme D150E/I168A, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.19
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.24
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225V
0.28
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q128A
0.29
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225A
0.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutants Q128K and Q225R
0.33
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.0, 25C, recombinant wild-type enzyme
0.33
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q128E
0.34
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225D
0.39
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225K
0.4
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant enzyme
0.4
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant wild-type enzyme
0.45
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant wild-type enzyme
0.46
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme I168A, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.5
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme D150E, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.52
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, native enzyme
0.52
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, native wild-type enzyme
0.54
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.55
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/L155A/G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.63
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225E
0.63
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
wild type enzyme, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.69
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/L155A, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.83
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L155A/G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.94
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/G8C/N60C/S65P, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
1.29
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L155A, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.7
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme D150E, at pH 7.5, at 25C
0.8
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme I168A, at pH 7.5, at 25C
1
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme F114A, at pH 7.5, at 25C; wild type enzyme, at pH 7.5, at 25C
1.3
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme N227H, at pH 7.5, at 25C
2.1
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme D150A, at pH 7.5, at 25C; mutant enzyme D150W, at pH 7.5, at 25C
49
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme F114A, at pH 7.5, at 25C
-
54
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme D150W, at pH 7.5, at 25C
-
57
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme D150A, at pH 7.5, at 25C
-
60
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme D150E, at pH 7.5, at 25C
-
62
-
N-carbobenzoxy-L-aspartic acid
-
wild type enzyme, at pH 7.5, at 25C
-
70
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme N227H, at pH 7.5, at 25C
-
105
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme I168A, at pH 7.5, at 25C
-
0.77
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
0.62
-
Phe-Leu-Ala-NH(CH2)2NH-dansyl
-
-
0.2
-
[3H]Tyr-Gly-Gly-Phe-Leu
-
pH 8.0, 37C
1.985
-
leucine enkephalin
-
pH 7.4, 37C, mutant D226A
additional information
-
additional information
-
effect of various n-pentanal concentrations of Km
-
additional information
-
additional information
-
-
-
additional information
-
additional information
-
overview: Km of synthetic oligopeptides
-
additional information
-
additional information
-
kinetics and thermodynamics of wild-type and mutant enzymes in absence or presence of salt, overview
-
additional information
-
additional information
-
thermodynamics of wild-type and mutant enzymes
-
additional information
-
additional information
-
kinetics
-
additional information
-
additional information
-
Michaelis-Menten kinetics, overview
-
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
2.36
-
(7-methoxycoumarin-4yl) acetyl-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH2
-
pH and temperature not specified in the publication
2.6
-
(europium(III) complex of a modified terpyridine)-K1K2K2GFSAK2K-black hole quencher 2
-
pH and temperature not specified in the publication
-
861
951
Benzyloxycarbonyl-(4-nitro)Phe-Leu-Ala
-
depending on mode of preparation of the form of enzyme
407
968
Benzyloxycarbonyl-Gly-Phe-Leu-Ala
-
depending on mode of preparation of the form of enzyme
362
605
Benzyloxycarbonyl-Phe-Leu-Ala
-
depending on mode of preparation of the form of enzyme
1.4
1.7
Dansyl-Gly-Phe-Ala
-
depending on assay method
0.06
-
GFA
-
pH 8.0, 37C
0.076
-
GFA
-
pH 8.0, 37C, 5 M glycerol
0.006
-
GFL
-
pH 8.0, 37C, 5 M glycerol, product GF
0.013
-
GFL
-
pH 8.0, 37C, product GF
0.026
-
GFL
-
pH 8.0, 37C, 5 M glycerol, product FL
0.045
-
GFL
-
pH 8.0, 37C, product FL
0.035
-
GFS
-
pH 8.0, 37C
0.05
-
GFS
-
pH 8.0, 37C, 5 M glycerol
0.07
-
GFSA
-
pH 8.0, 37C, product FSA
0.173
-
GFSA
-
pH 8.0, 37C, 5 M glycerol, product FSA
0.3
-
GFSA
-
pH 8.0, 37C, product GF
0.591
-
GFSA
-
pH 8.0, 37C, 5 M glycerol, product GF
6.08
-
GFSA
-
pH 8.0, 37C, 5 M glycerol, product GF
0.3
-
L-phenylalanine methyl ester
-
mutant enzyme F114A, at pH 7.5, at 25C
1.3
-
L-phenylalanine methyl ester
-
wild type enzyme, at pH 7.5, at 25C
2.1
-
L-phenylalanine methyl ester
-
mutant enzyme D150A, at pH 7.5, at 25C
2.3
-
L-phenylalanine methyl ester
-
mutant enzyme N227H, at pH 7.5, at 25C
2.5
-
L-phenylalanine methyl ester
-
mutant enzyme D150E, at pH 7.5, at 25C
3.2
-
L-phenylalanine methyl ester
-
mutant enzyme D150W, at pH 7.5, at 25C
4.9
-
L-phenylalanine methyl ester
-
mutant enzyme I168A, at pH 7.5, at 25C
0.083
-
leucine enkephalin
-
pH 6.8, 37C, mutant R203M
2.94
-
leucine enkephalin
-
pH 6.8, 37C, mutant D170A
2.94
-
leucine enkephalin
-
pH 7.4, 37C, mutant Y157F
155.5
-
leucine enkephalin
-
pH 7.4, 37C, wild-type
156
-
leucine enkephalin
-
pH 7.4, 37C, wild-type
1555
-
leucine enkephalin
-
pH 6.8, 37C, wild-type
0.556
-
N-(Benzyloxycarbonyl)-L-Phe
-
-
1.69
-
N-(Benzyloxycarbonyl)-L-Phe
-
-
13.58
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155A/I156V, pH 7.0, 37C
-
26.67
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155A/I156N, pH 7.0, 37C
-
48.33
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155S/I156N, pH 7.0, 37C
-
51.67
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant wild-type enzyme, pH 7.0, 37C
-
80
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155A, pH 7.0, 37C
-
105
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155S/I156V, pH 7.0, 37C
-
165
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155S, pH 7.0, 37C
-
4.8
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117R, pH 7.5, 25C
-
7.9
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant wild-type enzyme, pH 7.5, 25C
-
8.4
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117K, pH 7.5, 25C
-
10.5
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117E, pH 7.5, 25C
-
23.3
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
mutant L155F, 37C, pH 7.0
51.6
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
wild-type, 37C, pH 7.0
80
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
mutant L155A, 37C, pH 7.0
150
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
mutant L155G, 37C, pH 7.0
165
-
N-carbobenzoxy-Gly-L-Leu-NH2
-
mutant L155S, 37C, pH 7.0
1.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225A
1.6
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225V
3.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.0, 25C, recombinant mutant G8C/N60C/S65P
3.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225D
3.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/L155A/G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
3.4
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.0, 25C, recombinant wild-type enzyme
3.6
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q225R
3.7
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q128K
3.8
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant enzyme
3.8
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant wild-type enzyme
3.8
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutant Q128A
4.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, native enzyme
4.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, native wild-type enzyme
4.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant mutants Q128E and Q225E
4.7
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.5, 25C, recombinant wild-type enzyme and mutant Q225K
5.4
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
5.9
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
wild type enzyme, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
6.3
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L155A/G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
6.4
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/L155A, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
6.7
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.0, 25C, recombinant mutant L144S
7
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
pH 7.0, 25C, recombinant mutant G8C/N60C/S65P/L144S
7.4
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/G8C/N60C/S65P, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
7.7
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme I168A, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
8.7
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L155A, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
9.4
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
15.5
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme D150E/I168A, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
15.6
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S/D150E/S53D, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
16.5
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme D150E, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
17
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S/D150E, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
1.5
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme F114A, at pH 7.5, at 25C
9.5
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
wild type enzyme, at pH 7.5, at 25C
12.3
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme I168A, at pH 7.5, at 25C
17.1
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme N227H, at pH 7.5, at 25C
20.5
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme D150E, at pH 7.5, at 25C
24.4
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme D150A, at pH 7.5, at 25C
49.4
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme D150W, at pH 7.5, at 25C
0.3
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme F114A, at pH 7.5, at 25C
-
1.3
-
N-carbobenzoxy-L-aspartic acid
-
wild type enzyme, at pH 7.5, at 25C
-
2.1
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme D150A, at pH 7.5, at 25C
-
2.3
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme N227H, at pH 7.5, at 25C
-
2.5
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme D150E, at pH 7.5, at 25C
-
3.2
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme D150W, at pH 7.5, at 25C
-
4.9
-
N-carbobenzoxy-L-aspartic acid
-
mutant enzyme I168A, at pH 7.5, at 25C
-
6.9
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
1560
-
leucine enkephalin
-
pH 6.8, 37C, wild-type
additional information
-
additional information
-
effect of various n-pentanal concentrations of turnover number
-
additional information
-
additional information
-
overview: turnover number of synthetic oligopeptides
-
kcat/KM VALUE [1/mMs-1]
kcat/KM VALUE [1/mMs-1] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
1.03
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155A/I156V, pH 7.0, 37C
0
2
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155A/I156N, pH 7.0, 37C
0
3.67
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant wild-type enzyme, pH 7.0, 37C
0
4
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155A, pH 7.0, 37C; recombinant mutant L155S/I156N, pH 7.0, 37C
0
12
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155S/I156V, pH 7.0, 37C
0
12.67
-
N-benzyloxycarbonyl-Gly-L-Leu amide
-
recombinant mutant L155S, pH 7.0, 37C
0
9.6
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117R, pH 7.5, 25C
0
14.7
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117K, pH 7.5, 25C
0
18.8
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant wild-type enzyme, pH 7.5, 25C
0
23.9
-
N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
-
recombinant mutant G117E, pH 7.5, 25C
0
6
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/L155A/G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
6.7
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L155A, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
7.5
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L155A/G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
7.9
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/G8C/N60C/S65P, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
9.2
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme S53D/L155A, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
9.4
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
wild type enzyme, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
10
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme G8C/N60C/S65P, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
16.8
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme I168A, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
33.1
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme D150E, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
49.7
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
96.2
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S/D150E/S53D, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
99
-
N-carbobenzoxy-L-Asp-L-Phe methyl ester
-
mutant enzyme L144S/D150E, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
234387
1.5
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme F114A, at pH 7.5, at 25C
235706
9.3
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
wild type enzyme, at pH 7.5, at 25C
235706
11.7
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme D150A, at pH 7.5, at 25C
235706
13.7
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme N227H, at pH 7.5, at 25C
235706
16
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme I168A, at pH 7.5, at 25C
235706
23
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme D150W, at pH 7.5, at 25C
235706
27.6
-
N-carbobenzoxy-L-Asp-L-Phe-methyl ester
-
mutant enzyme D150E, at pH 7.5, at 25C
235706
0.23
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D/L155A/G8C/N60C/S65P, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
0.26
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L155A, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
0.29
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme G8C/N60C/S65P, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C; wild type enzyme, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
0.31
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D/G8C/N60C/S65P, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
0.32
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
0.33
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D/L155A, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
0.34
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L155A/G8C/N60C/S65P, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
0.62
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme I168A, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
0.79
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme D150E, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
1.82
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L144S, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
2.04
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L144S/D150E, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
2.1
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L155A, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
2.49
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L144S/D150E/S53D, in the absence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
3
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme I168A, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
3.2
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D/L155A, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
3.3
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D/L155A/G8C/N60C/S65P, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
3.5
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme G8C/N60C/S65P, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
3.8
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
wild type enzyme, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
3.9
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
4
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme D150E, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C; mutant enzyme S53D/G8C/N60C/S65P, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
4.3
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L155A/G8C/N60C/S65P, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
6
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L144S/D150E, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
6.6
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L144S, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
9
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme S53D, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
11.7
-
N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
-
mutant enzyme L144S/D150E/S53D, in the presence of 4 M NaCl, in 40 mM HEPES-NaOH buffer at pH 7.5 containing 10 mM CaCl2, at 25C
235751
15
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide
-
recombinant mutant G117R, pH 7.5, 25C
208784
16
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide
-
recombinant mutant G117K, pH 7.5, 25C
208784
33
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide
-
recombinant mutant G117E, pH 7.5, 25C
208784
40
-
N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide
-
recombinant wild-type enzyme, pH 7.5, 25C
208784
Ki VALUE [mM]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.000000064
-
1-beta-D-arabinofuranosyl-N4-lauroylcytosine
-
25C, pH 7.5
50
-
1-butanol
-
pH 7.5, 25C
460
-
1-butanol
-
pH 7.5, 25C
35
-
1-Pentanol
-
pH 7.5, 25C
38
-
1-Propanol
-
pH 7.5, 25C
65
-
1-Propanol
-
pH 7.5, 25C
280
-
1-Propanol
-
pH 7.5, 25C
0.0593
-
2-(4-methylphenyl)-3-(1,3-thiazol-2-yl) quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.57
-
2-(acetyloxy)-3-chlorobenzoic acid
-
in 100 mM Tris/HCl, pH 7.5, 2 mM CaCl2, 4% (v/v) DMSO, at 25C
-
40
-
2-butanol
-
pH 7.5, 25C
370
-
2-butanol
-
pH 7.5, 25C
0.315
-
2-ethyl-3-hydroxyquinazolin-4(3H)-one
-
in 100 mM Tris/HCl, pH 7.5, 2 mM CaCl2, 4% (v/v) DMSO, at 25C
-
0.00125
-
2-ethylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
2.06
-
2-hydroxy-N-(4-methyl-2-nitrophenyl)-3-nitrobenzamide
-
25C, pH 7.5
41
-
2-methyl-1-propanol
-
pH 7.5, 25C
380
-
2-methyl-1-propanol
-
pH 7.5, 25C
1.6
-
2-phenylpropionyl-L-phenylalanine
-
pH 8.6, temperature not specified in the publication
-
0.000015
-
2-phenylpropionyl-Leu-Trp
-
pH 7.2, temperature not specified in the publication
-
0.000243
-
3-(isopropylideneamino)-2,2-dimethyl-2,3-dihydroquinazolin-4(1H)-one
-
at pH 7.0 and 25C
-
0.0183
-
3-([(1E)-[4-(dimethylamino) phenyl]methylene]amino)-2-methylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.042
-
3-([(1E)-[4-(dimethylamino) phenyl]methylene]amino)-2-phenylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
10100
-
3-amino-2-(4-chlorophenyl)quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
3.12
-
3-amino-2-(4-nitrophenyl)quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.0379
-
3-amino-2-(trifluoromethyl) quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.0549
-
3-amino-2-methylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.128
-
3-hydroxy-2-isopropylquinazolin-4(3H)-one
-
in 100 mM Tris/HCl, pH 7.5, 2 mM CaCl2, 4% (v/v) DMSO, at 25C
-
0.93
-
3-hydroxy-2-methylquinazolin-4(3H)-one
-
in 100 mM Tris/HCl, pH 7.5, 2 mM CaCl2, 4% (v/v) DMSO, at 25C
-
1.7
-
3-methylaspirin
-
in 100 mM Tris/HCl, pH 7.5, 2 mM CaCl2, 4% (v/v) DMSO, at 25C
-
0.0000115
-
3-phenyl-2-(trifluoromethyl) quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
4
-
3-[[(1E)-(3-chlorophenyl)methylene]amino]-2-phenylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
4.81
-
5-bromo-N-(4-bromophenyl)-2-hydroxy-3-nitro-benzamide
-
25C, pH 7.5
0.0016
-
CGS-28,106
-
pH 8.0, 37C
7.63
-
Dimethylformamide
-
pH 7.5, 25C
7.7
-
DMSO
-
pH 7.5, 25C
106
-
ethanimidic acid N-[4-oxo-2-phenyl-3(4H)-quinazolinyl]-ethyl ester
-
at pH 7.0 and 25C
-
100
-
ethanol
-
pH 7.5, 25C
0.0031
-
HACBO-Gly
-
pH 7.4, 37C, wild-type
0.019
-
HACBO-Gly
-
pH 7.4, 37C, mutant D226A
0.031
-
HACBO-Gly
-
pH 7.4, 37C, mutant Y157F
0.25
-
Isopropanol
-
pH 7.5, 25C
294
-
N-(2,3-dimethylphenyl)-2-hydroxy-3-nitro-benzamide
-
25C, pH 7.5
161
-
N-(2,4-dimethylphenyl)-2-hydroxy-3-nitro-benzamide
-
25C, pH 7.5
1.45
-
N-(2,5-dimethylphenyl)-2-hydroxy-3-nitrobenzamide
-
25C, pH 7.5
697
-
N-(2-chloro-4-nitrophenyl)-2-hydroxy-3-nitro-benzamide
-
25C, pH 7.5
0.047
-
N-(2-chloro-6-methylphenyl)-2-hydroxy-3-nitrobenzamide
-
25C, pH 7.5
2.95
-
N-(2-ethylphenyl)-2-hydroxy-3-nitrobenzamide
-
25C, pH 7.5
8.14
-
N-(5-chloro-2-methoxyphenyl)-2-hydroxy-3-nitro-benzamide
-
25C, pH 7.5
0.008
-
N-Benzyloxycarbonyl-L-phenylalanine
-
pH 5.0, 37C, Co2+-bound enzyme
0.012
-
N-Benzyloxycarbonyl-L-phenylalanine
-
pH 5.1, 37C, Zn2+-bound enzyme
0.16
-
N-Benzyloxycarbonyl-L-phenylalanine
-
pH 7.1, 37C, Co2+-bound enzyme
0.37
-
N-Benzyloxycarbonyl-L-phenylalanine
-
pH 7.0, 37C, Zn2+-bound enzyme
0.007
-
N-benzyloxycarbonyl-L-tryptophan
-
pH 5.0, 37C, Co2+-bound enzyme
-
0.008
-
N-benzyloxycarbonyl-L-tryptophan
-
pH 5.0, 37C, Zn2+-bound enzyme
-
0.087
-
N-benzyloxycarbonyl-L-tryptophan
-
pH 7.0, 37C, Zn2+-bound enzyme
-
0.19
-
N-benzyloxycarbonyl-L-tryptophan
-
pH 7.0, 37C, Zn2+-bound enzyme, in presence of 1 M NaSCN
-
0.21
-
N-benzyloxycarbonyl-L-tryptophan
-
pH 7.0, 37C, Co2+-bound enzyme, in presence of 1 M NaSCN
-
0.18
-
n-Propanol
-
pH 7.5, 25C
0.214
-
oxyquinoline
-
25C, pH 7.5
0.00003
-
phosphoramidon
-
pH 7.2, temperature not specified in the publication
0.000031
-
phosphoramidon
-
pH 7.4, 37C, mutant Y157F
0.000032
-
phosphoramidon
-
pH 7.2, 25C
0.00006
-
phosphoramidon
-
pH 6.8, 37C, wild-type
0.00006
-
phosphoramidon
-
pH 7.4, 37C, wild-type
0.00019
-
phosphoramidon
-
pH 7.4, 37C, mutant D226A
0.0047
-
phosphoramidon
-
pH 6.8, 37C, mutant R203M
0.0008
-
Retrothiorphan
-
pH 6.8, 37C, wild-type
0.16
-
Retrothiorphan
-
pH 6.8, 37C, mutant R203M
110
-
tert-amylalcohol
-
pH 7.5, 25C
190
-
tert-butyl alcohol
-
pH 7.5, 25C
0.0016
-
thiorphan
-
pH 6.8, 37C, wild-type
0.0016
-
thiorphan
-
pH 8.0, 37C
0.0016
-
thiorphan
-
pH 7.4, 37C, wild-type
0.0033
-
thiorphan
-
pH 6.8, 37C, mutant D170A
0.0076
-
thiorphan
-
pH 7.4, 37C, mutant Y157F
0.013
-
thiorphan
-
pH 7.4, 37C, mutant D226A
0.16
-
thiorphan
-
pH 6.8, 37C, mutant R203M
0.000012
-
Zincov
-
pH 7.5, 25C, cobalt-substituted thermolysin
0.00023
-
Zincov
-
pH 7.5, 25C
0.0012
-
[(2S)-2-sulfanyl-3-phenylpropanoyl]Gly-(5-Ph)Pro
-
pH 8.0, 37C
0.000042
-
[(2S)-2-sulfanyl-3-phenylpropanoyl]Phe-Tyr
-
pH 8.0, 37C
0.000048
-
[(2S,R)-2-sulfanylheptanoyl]Phe-Ala
-
pH 8.0, 37C
430
-
methanol
-
pH 7.5, 25C
additional information
-
additional information
-
detailed inhibition kinetics with Co2+ and Zn2+, overview
-
additional information
-
additional information
-
inhibition kinetics with Co2+ and Ca2+, overview
-
additional information
-
additional information
-
organic solvent inhibition kinetics, overview
-
IC50 VALUE [mM]
IC50 VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.07685
-
2-(4-methylphenyl)-3-(1,3-thiazol-2-yl) quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.00125
-
2-ethylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.0002477
-
3-(isopropylideneamino)-2,2-dimethyl-2,3-dihydroquinazolin-4(1H)-one
-
at pH 7.0 and 25C
-
0.01832
-
3-([(1E)-[4-(dimethylamino) phenyl]methylene]amino)-2-methylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.04203
-
3-([(1E)-[4-(dimethylamino) phenyl]methylene]amino)-2-phenylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
12.74
-
3-amino-2-(4-chlorophenyl)quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
3.118
-
3-amino-2-(4-nitrophenyl)quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.03786
-
3-amino-2-(trifluoromethyl) quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.05489
-
3-amino-2-methylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
0.0000115
-
3-phenyl-2-(trifluoromethyl) quinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
4.002
-
3-[[(1E)-(3-chlorophenyl)methylene]amino]-2-phenylquinazolin-4(3H)-one
-
at pH 7.0 and 25C
-
122.6
-
ethanimidic acid N-[4-oxo-2-phenyl-3(4H)-quinazolinyl]-ethyl ester
-
at pH 7.0 and 25C
-
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.34
-
-
mutant F114H, substrate casein, 25C, pH 7.5
0.42
-
-
mutant I168H, substrate casein, 25C, pH 7.5; mutant S234A, substrate casein, 25C, pH 7.5
0.55
-
-
mutant N227A, substrate casein, 25C, pH 7.5
0.62
-
-
mutant D150W, substrate casein, 25C, pH 7.5
0.68
-
-
mutant I168A, substrate casein, 25C, pH 7.5
0.84
-
-
mutant D150H, substrate casein, 25C, pH 7.5
0.89
-
-
mutant D150A, substrate casein, 25C, pH 7.5; mutant S169A, substrate casein, 25C, pH 7.5
0.93
-
-
mutant D150E, substrate casein, 25C, pH 7.5
1.08
-
-
wild-type, substrate casein, 25C, pH 7.5
1.14
-
-
mutant F114A, substrate casein, 25C, pH 7.5
1.18
-
-
mutant N227H, substrate casein, 25C, pH 7.5
3.5
-
-
purified mutant enzyme L144S/D150E, using casein as substrate, at pH 7.5 and 25C
3.6
-
-
purified mutant enzyme L144S, using casein as substrate, at pH 7.5 and 25C
6.7
-
-
purified mutant enzyme L155A, using casein as substrate, at pH 7.5 and 25C
7.5
-
-
purified mutant enzyme D150E/I168A, using casein as substrate, at pH 7.5 and 25C
7.9
-
-
purified mutant enzyme S53D/L155A/G8C/N60C/S65P, using casein as substrate, at pH 7.5 and 25C
8.1
-
-
purified mutant enzyme L155A/G8C/N60C/S65P, using casein as substrate, at pH 7.5 and 25C
9.6
-
-
purified mutant enzyme I168A, using casein as substrate, at pH 7.5 and 25C
10.2
-
-
purified mutant enzyme S53D/L155A, using casein as substrate, at pH 7.5 and 25C
10.5
-
-
purified mutant enzyme D150E, using casein as substrate, at pH 7.5 and 25C
10.8
-
-
purified mutant enzyme G8C/N60C/S65P, using casein as substrate, at pH 7.5 and 25C; purified wild type enzyme, using casein as substrate, at pH 7.5 and 25C
11.3
-
-
purified mutant enzyme S53D, using casein as substrate, at pH 7.5 and 25C
11.9
-
-
purified mutant enzyme S53D/G8C/N60C/S65P, using casein as substrate, at pH 7.5 and 25C
2000
-
-
recombinant mutant G117D, substrate casein, pH 7.5, 25C
2900
-
-
recombinant mutant G117R, substrate casein, pH 7.5, 25C
3200
-
-
recombinant mutant G117K, substrate casein, pH 7.5, 25C
9500
-
-
pH 7.5, 25C
11000
-
-
recombinant wild-type enzyme, substrate casein, pH 7.5, 25C
12000
-
-
recombinant mutant G117E, substrate casein, pH 7.5, 25C
additional information
-
-
catalytic efficiency of wild-type and mutant enzymes in absence or presence of 4 M NaCl, overview
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.8
-
-
F-AspPheOMe as substrate
7
7.5
Bacillus proteolyticus
-
-
7
7.5
-
assay at
7
-
-
around pH 7.0
7
-
-
assay at
7.3
-
-
Z-ArgPheOMe as substrate
7.4
-
-
assay at
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.5
8.5
-
for hydrolysis of neutral substrate N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, wild-type and mutants N112D, N112E exhibit bell-shaped pH-dependence. For hydrolysis of negatively charged substrate N-carbobenzoxy-L-Asp-L-Phe methyl ester, wild-type shows bell-shaped pH-dependence, for mutants N112D and N112E, pH-dependence of the ratio kcat/Km decreases with increase in pH from 5.5 to 8.5
additional information
-
-
pH profile
additional information
-
-
bell-shaped pH-dependence with importance of surfacecharges of thermolysin, the bell-shaped pH dependence profile of the FAGLA-hydrolyzing activity of thermolysin is shifted from pH 5.4 to pH 6.7 by the addition of 4 M NaCl
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
37
-
-
assay at
37
-
-
assay at
50
-
Bacillus proteolyticus
-
-
TEMPERATURE RANGE
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
70
-
hydrolysis of bovine alpha-lactalbumin at 25C and 70C under nonreducing conditions. At 25C, substrate undergoes limited hydrolysis leading to peptides no longer degraded. At 70C, protein is first quickly cleaved, then unfolded, leading to the release of intermediate peptides that may be further degraded
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
from newborn foreskin
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
secretion from cytosol via periplasmic space
Manually annotated by BRENDA team
Geobacillus stearothermophilus MK232
-
secretion from cytosol via periplasmic space
-
Manually annotated by BRENDA team
-
secretion mechanism, overview
-
Manually annotated by BRENDA team
-
secretion from cytosol via periplasmic space
-
Manually annotated by BRENDA team
-
supplementation of growth medium by calcium induces expression. Regulation by calcium ions is at posttranscriptional level
-
Manually annotated by BRENDA team
Bacillus thermoproteolyticus Rokko
-
-
-
-
Manually annotated by BRENDA team
-
pro-enzyme form, folding and autocleavage takes place in the periplasmic space, overview
-
Manually annotated by BRENDA team
-
secretion from cytosol via periplasmic space
-
Manually annotated by BRENDA team
Geobacillus stearothermophilus MK232
-
secretion from cytosol via periplasmic space
-
-
Manually annotated by BRENDA team
Geobacillus stearothermophilus MK232
-
secretion from cytosol via periplasmic space
-
-
Manually annotated by BRENDA team
additional information
-
the protein is translocated through the membrane at the expense of ATP and the pre-peptide is cleaved off by a type I signal peptidase
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
34600
-
-
gel filtration
34800
-
-
Bacillus thermoproteolyticus, amino acid composition, amino acid sequence
36000
-
-
Western blot
36400
-
-
gel filtration
37500
-
-
Bacillus thermoproteolyticus, sedimentation equilibrium method
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 34600, SDS-PAGE
?
-
x * 22 852, propeptide of thermolysin, sequence calculation, x * 26000, mature enzyme, SDS-PAGE
?
-
x * 35000, SDS-PAGE, x * 55170, calculated
monomer
-
1 * 37000-38000, Bacillus thermoproteolyticus, SDS-PAGE, gel filtration in 6 M guanidine hydrochloride
additional information
-
in presence of Ca2+, enzyme exhibits four autodegradation sites, one of them being Gly154-Leu155
additional information
-
the enzyme consists of a beta-rich N-terminal domain and an alpha-helix C-terminal domain connected by a central alpha-helix, which is located at the bottom of the active site cleft
additional information
-
structure determination and molecular dynamics simulations, overview
additional information
-
TLPs have a two domain structure
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
proteolytic modification
-
synthesis as a pre-proprotein
proteolytic modification
-
the pre-pro-enzyme contains a signal peptide and a prosequence, the prosequence acts as an intramolecular chaperone for autocatalytic cleavage of the linking peptide bond
proteolytic modification
-
thermolysin performs Co2+-stimulable autolysis
proteolytic modification
-
thermolysin is synthesized as inactive pre-proenzyme and receives autocatalytic cleavage of the peptide bond linking the pro- and mature sequences, overview
proteolytic modification
-
the pre-pro-enzyme contains a signal peptide and a prosequence, the prosequence acts as an intramolecular chaperone for autocatalytic cleavage of the linking peptide bond
proteolytic modification
-
autolytic processing. Secreted proteases are produced as prepro-proteins. The pre-peptide is cleaved-off during Sec-controlled secretion, and the active protease emerges outside the cell after folding of the proprotein and autolytic removal of the pro-peptide. The protein is translocated through the membrane at the expense of ATP and the pre-peptide is cleaved off by a type I signal peptidase. The pro-part plays two roles in this process: it facilitates folding by acting as an intra-molecular chaperone and it inhibits protease activity of the folded pro-enzyme
proteolytic modification
Geobacillus stearothermophilus MK232
-
the pre-pro-enzyme contains a signal peptide and a prosequence, the prosequence acts as an intramolecular chaperone for autocatalytic cleavage of the linking peptide bond
-
proteolytic modification
-
autodegradation at position 154-155
additional information
-
no glycoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
commercial preparation of crystalline enzyme
-
crystal structure determination
-
crystallization of tetragonal thermolysin, at room temperature, structure determination and anaylsis, fluctuations and solvent-accessible surface areas, biological nanopores and water densities, and radial distributions of water and ions, overview
-
data in presence of 4 M NaCl, introduced into crystals originally grown without NaCl
-
multiple-solvent crystal structure determination cell dimensions dependent on isopropanol concentration
-
native thermolysin in complex with 3-methylaspirin, 3-methylaspirin ethyl ester, and 3-methylaspirin cyclopropyl ester, sitting drop vapor diffusion method, using water as reservoir solution, at 18C
-
recrystallized 3 times from DMSO
-
resiudes H142, H146, and E166 coordinate the catalytic zinc, while E143 and H231 are required for the catalytic activity
-
structure, electron density map at 2.3 A resolution
-
thiocyanate as crystallizing agent, space group P6(1)22, unit-cell parameters a and b : 93.17 A
-
three times crystallized and lyophilized preparation, Lot T5CB491
-
three-times crystallized preparation, Lot T5CB491 from Daiwa Kasei, Osaka
-
TLN-inhibitor complex, 3 alpha-mercaptoacyldipeptides in the thermolysin active site crystallized, hexagonal crystals, space group P6(1)22, [(2S)-2-sulfanyl-3-phenylpropanoyl]Gly-(5-Ph)Pro, parameters a : b : 93.50 A, c : 131.30 A, [(2S)-2-sulfanyl-3-phenylpropanoyl]Phe-Tyr, a : b : 93.97 A, c : 131.79 A, [(2S,R)-2-sulfanylheptanoyl]Phe-Ala, a : b : 93.31 A, c : 131.83 A
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
7
-
-
after precipitation with trichloroacetic acid the purified enzyme irreversibly loses activity and solubility at neutral pH
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
37
-
-
incubation of impure enzyme at 37C causes proteolysis of impurities, whereas the protease remains active and uncleaved
80
-
-
1 h, heated enzyme has the same maximum velocity but lower affinity for substrate than the native
80
-
-
thermoinactivation of thermolysin at 80C in 50% of solvents, half-lives of 3-20 min, with the increase in solvent hydrophobicity, thermal stability of the enzyme decreases, overview
80
-
-
the rate constant (kobs) for thermal inactivation at 80C is 84000 s-1 for the wild type enzyme
80
-
-
half-life of wild-type enzyme is 18.3 min, half-lives of mutants are 25 min for L155A, 24 min for L155S, 60.8 min for L155A/I156V, 62.4 min for L155S/I156N, 93.3 for L155S/I156N, and 40 min for L155S/I156V
82
-
-
the enzyme loses 50% of its activity after 30 min at 82C
85
-
-
30 min, 51% remaining activity of recombinaqnt wild-type enzyme, 35-78% remaining activity of mutant enzymes
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
After precipitation with trichloroacetic acid the purified enzyme irreversibly loses activity and solubility at neutral pH
-
Ca2+ increases thermal stability
-
Calcium is necessary to stabilize the structure of thermolysin
-
enzyme is stable in presence of CaCl2
-
ORGANIC SOLVENT
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
dimethylformamide
-
half-life is 6 min at 80C, trehalose stabilizes the enzyme in DMF
DMSO
-
half-life is over 20 min at 80C
n-propanol
-
half-life is 3 min at 80C, glycerol stabilizes the enzyme
isopropanol
-
half-life is 4 min at 80C, stabilizes the enzyme
additional information
-
solvents cause mixed inhibition of thermolysin, kinetic and structural studies, overview
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant extracellular wild-type and mutant enzymes from Bacillus subtilis culture medium by ammonium sulfate fractionation, hydrophobic interaction chromatography, and gel filtration
-
by affinity chromatography
-
by Gly-D-Phe affinity chromatography, coupling to the resin by epichlorohydrin, 1,4-butandiol diglycidyl ether, or 1,6-hexanediol diglycidyl ether, method optimization and evaluation, overview
-
commercial enzyme powder further purified to remove salts
-
commercial preparation, used without further purification
-
evaluation of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands for thermolysin, each of the ligands is immobilized to a resin. The optimum pH for adsorption of thermolysin is pH 5.0 to pH 6.0 for each of the ligands, affinity chromatography method development, adsorption isotherms, overview
-
expression as single polypeptide pre-proenzyme in Escherichia coli, secretion into medium as mature enzyme
-
mobile phase effects in the high-performance affinity purification
-
recombinant wild-type and mutant enzymes from Escherichia coli K12 strain JM109 by hydrophobic interaction and Gly-D-Phe affinity chromatography
-
recombinant wild-type and mutant enzymes from Escherichia coli K12 strain JM109 to homogeneity by Gly-D-Phe heat treatment at 60C for 20 min, affinity chromatography and gel filtration
-
recombinant wild-type and mutated thermolysin
-
wild-type and mutated thermolysin
-
recombinant enzyme, different methods, e.g. by Gly-D-Phe or bacitracin affinity, ion exchange, and hydrophobic interaction chromatography, gel filtration and ammonium sulfate fractionation, detailed overview
-
incubation of impure enzyme at 37C causes proteolysis of impurities, whereas the protease remains active and uncleaved
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expression of wild-type and mutant enzymes in Bacillus subtilis by vector pTB51, the enzymes are secreted to the culture medium
-
cloned in Bacillus subtilis DB117, plasmids subcloned in Escherichia coli
-
expressed in Escherichia coli strain K12 JM109
-
expression of wild-type and mutant enzymes in Escherichia coli K12 strain JM109
-
expression of wild-type and mutant enzymes in Escherichia coli K12 strain JM109 and secretion to the cell culture medium
-
expression of wild-type and mutant enzymes in Escherichia coli strain T5KC991
-
gene npr, DNA and amino acid sequence determination, expression of wild-type and mutant enzymes in Escherichia coli K12 strain JM109
-
gene npr, expression of wild-type and mutant enzymes in Escherichia coli K12 strain JM109
-
genes npr and nprT, DNA and amino acid sequence determination and analysis, expression in Escherichia coli and Bacillus subtilis
-
genes npr and nprT, DNA and amino acid sequence determination and analysis, expression in Escherichia coli and Bacillus subtilis, different methods, expression of tagged mature enzyme, proenzyme, or mutant enzyme in inclusion bodies or in the cytosol or medium as soluble protein, overview
-
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
L155A
-
site-directed mutagenesis, the mutation abolishes the autodegradation activity, mutant thermostability at 80C is slightly enhanced compared to the wild-type enzyme
L155A/I156N
-
site-directed mutagenesis, the mutation abolishes the autodegradation activity, mutant thermostability at 80C is enhanced compared to the wild-type enzyme
L155A/I156V
-
site-directed mutagenesis, the mutation abolishes the autodegradation activity, mutant thermostability at 80C is enhanced compared to the wild-type enzyme
L155S
-
site-directed mutagenesis, the mutation abolishes the autodegradation activity, mutant thermostability at 80C is slightly enhanced compared to the wild-type enzyme
L155S/I156N
-
site-directed mutagenesis, the mutation abolishes the autodegradation activity, mutant thermostability at 80C is enhanced compared to the wild-type enzyme
L155S/I156V
-
site-directed mutagenesis, the mutation abolishes the autodegradation activity, mutant thermostability at 80C is enhanced compared to the wild-type enzyme
A113D
-
complete loss of activity
A113E
-
complete loss of activity
A113H
-
complete loss of activity
A113K
-
complete loss of activity
A113R
-
complete loss of activity
A4T/G8C/T56A/G58A/N60C/T63F/S65P/A69P
-
the mutant shows altered thermodynamics
A4T/T56A/G58A/T63F/S65P/A69P
-
the mutant shows altered thermodynamics
D150A
-
131% residual activity with casein, 81% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
D150A
-
mutant with improved activity, the mutant has higher kcat values in N-carbobenzoxy-L-Asp-L-Phe-methyl ester synthesis than wild type
D150E
-
128% residual activity with casein, 228% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
D150E
-
mutant with improved activity, the mutant has higher kcat values in N-carbobenzoxy-L-Asp-L-Phe-methyl ester synthesis than wild type
D150E
-
the mutation increases the activity of thermolysin
D150H
-
105% residual activity with casein, 37% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide. Thermal inactivation at 80C is greatly suppressed
D150K
-
51% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
D150R
-
44% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
D150W
-
81% residual activity with casein, 60% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide. Thermal inactivation at 80C is greatly suppressed
D150W
-
mutant with improved activity, the mutant has higher kcat values in N-carbobenzoxy-L-Asp-L-Phe-methyl ester synthesis than wild type
D170A
-
site-directed mutagenesis
D170A
-
complete loss of activity
D170E
-
complete loss of activity
D170H
-
complete loss of activity
D170K
-
complete loss of activity
D170R
-
complete loss of activity
D226A
-
site-directed mutagenesis
DELTA127
-
absence of CaCl2, 18% of wild-type activity, presence of 5 mM CaCl2, 71% of wild-type activity. Decrease in amount of enzyme secreted compared to wild-type
E143A
-
site-directed mutagenesis, E143A might exist as a complex with the propetide in the supernatant, inactive mutant, the autocatalytic activity is affected
E143R
-
inactive mutant
E143S
-
inactive mutant
E143W
-
inactive mutant
F114A
-
28% residual activity with casein, 8% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
F114A
-
mutant with reduced activity
F114D
-
complete loss of activity
F114E
-
complete loss of activity
F114H
-
18% residual activity with casein, 20% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
F114K
-
complete loss of activity
F114R
-
complete loss of activity
G117D
-
site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type
G117E
-
site-directed mutagenesis, the mutant enzyme shows increased activity compared to the wild-type enzyme, the kcat/Km value is 80% of wild-type level with N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but 130% with N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
G117K
-
site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type, the kcat/Km value is 40% of wild-type level with N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide, but 80% with N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
G117R
-
site-directed mutagenesis, the mutant enzyme shows reduced activity compared to the wild-type, the kcat/Km value is 40% of wild-type level with N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-benzyloxycarbonyl-L-Asp-L-Phe-methyl ester
G162A
-
complete loss of activity
G162D
-
complete loss of activity
G162E
-
complete loss of activity
G162H
-
complete loss of activity
G162K
-
complete loss of activity
G162R
-
complete loss of activity
G8C/N60C
-
the mutant shows altered thermodynamics
G8C/N60C/S65P
-
site-directed mutagenesis, the mutant shows a similar catalytic efficiency compared tot he wild-type enzyme
G8C/N60C/S65P
-
the mutation increases the stability of thermolysin; the triple mutation increases the stability of thermolysin as high as the individual mutations do
G8C/N60C/S65P/L144S
-
site-directed mutagenesis, the mutant shows about 6fold increased catalytic effiency compared to the wild-type enzyme
G8C/N60C/S65P/L144S
-
the mutant is more active and stable than wild type thermolysin
H231A
-
the mutant shows 500fold decreased catalytic efficiency compared to the wild-type enzyme
I168A
-
69% residual activity with casein, 125% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide. Thermal inactivation at 80C is greatly suppressed
I168A
-
mutant with improved activity, the mutant has higher kcat values in N-carbobenzoxy-L-Asp-L-Phe-methyl ester synthesis than wild type
I168A
-
the mutation increases the activity of thermolysin and shows about 90% casein-hydrolytic activity compared to the wild type enzyme
I168D
-
complete loss of activity
I168E
-
complete loss of activity
I168H
-
13% residual activity with casein, 35% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide. Thermal inactivation at 80C is greatly suppressed
I168K
-
complete loss of activity
I168R
-
complete loss of activity
L144S
-
site-directed mutagenesis, the mutant shows about 10fold increased catalytic effiency compared to the wild-type enzyme
L144S
-
the mutation increases the activity of thermolysin and shows about 30% casein-hydrolytic activity compared to the wild type enzyme
L144S/D150E
-
the mutation yields the most significant increase in the hydrolytic activities for N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide and N-carbobenzoxy-L-Asp-L-Phe methyl ester and shows about 30% casein-hydrolytic activity compared to the wild type enzyme
L144S/D150E/I168A/S53D/L155A/G8C/N60C/S65P
-
inactive
L144S/D150E/L155A
-
inactive
L144S/D150E/S53D
-
the triple mutant shows improved activity and stability with about 30% casein-hydrolytic activity compared to the wild type enzyme
L144S/D150E/S53D/L155A
-
inactive
L144S/D150W/N227H
-
the mutant shows 10fold decreased catalytic efficiency compared to the wild-type enzyme
L144S/I168A
-
the mutation abolishes the hydrolytic activities for N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide and N-carbobenzoxy-L-Asp-L-Phe methyl ester
L155A
-
thermostability at 80C increases with amino acid substitutions at L155 in the order of wild-type, Gly, Ser, Phe, Ala. Autodegradation site shifts from G154-L155 to A155-I156 and the bond I164-D165 is newly recognized as an autodegradation site
L155A
-
absence of CaCl2, 87% of wild-type activity, presence of 5 mM CaCl2, 83% of wild-type activity. Amount of enzyme secreted is about the same level as wild-type
L155A
-
the mutation increases the stability of thermolysin and shows about 60% casein-hydrolytic activity compared to the wild type enzyme
L155A/G8C/N60C/S65P
-
the mutant shows about 80% casein-hydrolytic activity compared to the wild type enzyme
L155F
-
thermostability at 80C increases with amino acid substitutions at L155 in the order of wild-type, Gly, Ser, Phe, Ala. Autodegradation site shifts from G154-L155 to F155-I156 and the bond I164-D165 is newly recognized as an autodegradation site
L155G
-
thermostability at 80C increases with amino acid substitutions at L155 in the order of wild-type, Gly, Ser, Phe, Ala. Autodegradation site shifts from G154-L155 to G155-I156 and the bond I164-D165 is newly recognized as an autodegradation site
L155S
-
thermostability at 80C increases with amino acid substitutions at L155 in the order of wild-type, Gly, Ser, Phe, Ala. Autodegradation site shifts from G154-L155 to S155-I156 and the bond I164-D165 is newly recognized as an autodegradation site
L155S
-
the mutant shows increased stability at 80C compared to the wild-type enzyme
N112A
-
no enzymic activity in supernatant of cells expressing mutant
N112A
-
site-directed mutagenesis, inactive mutant, the autocatalytic activity is affected
N112D
-
supernatants of cells expressing mutant show 18% of wild-type activity
N112D
-
the mutant shows an altered pKa value
N112D
-
site-directed mutagenesis, the autocatalytic activity is affected
N112E
-
supernatants of cells expressing mutant show 5% of wild-type activity
N112E
-
site-directed mutagenesis, the autocatalytic activity is affected
N112H
-
no enzymic activity in supernatant of cells expressing mutant
N112H
-
site-directed mutagenesis, inactive mutant, the autocatalytic activity is affected
N112K
-
no enzymic activity in supernatant of cells expressing mutant
N112K
-
site-directed mutagenesis, inactive mutant, the autocatalytic activity is affected
N112R
-
no enzymic activity in supernatant of cells expressing mutant
N112R
-
site-directed mutagenesis, inactive mutant, the autocatalytic activity is affected
N116D/Q119R/D150Q/Q225R
-
the mutant shows 4fold decreased catalytic efficiency compared to the wild-type enzyme
N227A
-
72% residual activity with casein , 28% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide. Thermal inactivation at 80C is greatly suppressed
N227D
-
11% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
N227E
-
36% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
N227H
-
19% residual activity with casein, 19% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide. Thermal inactivation at 80C is greatly suppressed
N227H
-
mutant with improved activity, the mutant has higher kcat values in N-carbobenzoxy-L-Asp-L-Phe-methyl ester synthesis than wild type
N227K
-
29% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
N227R
-
55% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
P208H
-
absence of CaCl2, 0.61% of wild-type activity, presence of 5 mM CaCl2, 61% of wild-type activity. Decrease in amount of enzyme secreted compared to wild-type
Q128A
-
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
Q128E
-
site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme
Q128K
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Q225A
-
site-directed mutagenesis, the mutant shows altered pKa value and stimulation of activity by NaCl and reduced activity with the negatively charged substrate N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester substrate compared to the wild-type enzyme
Q225D
-
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
Q225E
-
site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme
Q225K
-
site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme
Q225R
-
site-directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme
Q225V
-
site-directed mutagenesis, the mutant shows altered pKa value and stimulation of activity by NaCl and reduced activity with the negatively charged substrate N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester substrate compared to the wild-type enzyme
R203A
-
the mutant shows 5fold decreased catalytic efficiency compared to the wild-type enzyme
R203M
-
site-directed mutagenesis
R203M
-
the mutant shows 2300fold decreased catalytic efficiency compared to the wild-type enzyme
S103A
-
the mutant shows 3fold decreased catalytic efficiency compared to the wild-type enzyme
S169A
-
112% residual activity with casein, 64% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
S169D
-
complete loss of activity
S169E
-
complete loss of activity
S169H
-
complete loss of activity
S169K
-
complete loss of activity
S169R
-
complete loss of activity
S198D
-
site directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme
S218D
-
site directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme
S234A
-
88% residual activity with casein, 17% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide. Thermal inactivation at 80C is greatly suppressed
S234D
-
5% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
S234E
-
4% residual activity with casein, 7% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
S234H
-
32% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
S234K
-
complete loss of activity
S234R
-
complete loss of activity
S254D
-
site directed mutagenesis, the mutant shows similar activity compared to the wild-type enzyme
S25D
-
site directed mutagenesis, the catalytic activity is of the mutant enzyme is similar to the wild-type in absence of NaCl, but increased in presence of 4 M NaCl
S53D
-
site directed mutagenesis, the catalytic activity is of the mutant enzyme is similar to the wild-type in absence of NaCl, but increased in presence of 4 M NaCl, increased thermostability in presence of 10 mM CaCl2
S53D
-
the mutation increases the stability of thermolysin
S53D/G8C/N60C/S65P
-
the mutant shows about 110% casein-hydrolytic activity compared to the wild type enzyme
S53D/L155A
-
the mutation yields the greatest increase in the thermal stability and shows about 90% casein-hydrolytic activity compared to the wild type enzyme
S53D/L155A/G8C/N60C/S65P
-
the mutant shows about 70% casein-hydrolytic activity compared to the wild type enzyme
S65D
-
site directed mutagenesis, the catalytic activity is of the mutant enzyme is similar to the wild-type in absence of NaCl, but increased in presence of 4 M NaCl, increased thermostability in presence of 10 mM CaCl2
V230A
-
17% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
V230D
-
complete loss of activity
V230E
-
complete loss of activity
V230H
-
complete loss of activity
V230K
-
3% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
V230R
-
6% residual activity with casein, 12% residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
W115A
-
complete loss of activity
W115D
-
complete loss of activity
W115E
-
complete loss of activity
W115H
-
complete loss of activity
W115K
-
complete loss of activity
W115L
-
inactive mutant
W115R
-
complete loss of activity
W115V
-
inactive mutant
Y157A
-
11% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
Y157D
-
1% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
Y157E
-
13% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
Y157F
-
site-directed mutagenesis
Y157H
-
24% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
Y157K
-
7% residual activity with casein, no residual activity with substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide
Y157R
-
complete loss of activity
D170A
Bacillus thermoproteolyticus Rokko
-
site-directed mutagenesis
-
M205P
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absence of CaCl2, 0.52% of wild-type activity, presence of 5 mM CaCl2, 48% of wild-type activity. Decrease in amount of enzyme secreted compared to wild-type
additional information
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generation of an engineered enzyme with a higher activity in the synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester
additional information
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a mutant thermolysin is affected by its autocatalytic digestion activity
R203M
Bacillus thermoproteolyticus Rokko
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site-directed mutagenesis
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additional information
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generation of an engineered enzyme with a higher activity in the synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester
additional information
Geobacillus stearothermophilus MK232
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generation of an engineered enzyme with a higher activity in the synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester
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Renatured/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
solubilization and refolding of recombinant enzyme from inclusion bodies after expression in Escherichia coli
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APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
synthesis
Bacillus proteolyticus
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production of L-alpha-aspartame, which is used as a low-calorie sweetener in food, including soft drinks, table-top sweeteners, dairy products, instant mixes, dressings, jams, confectionary, toppings and in pharmaceuticals
analysis
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thermolysin degrades cellular prion protein while preserving both proteinase K-sensitive and proteinase K-resistant isoforms of disease-related prion protein in both rodent and human prion strains. In variant Creutzfeldt-Jakob disease, up to 90% of total prion protein present in the brain resists degradation with thermolysin, whereas only about 15% of this material resists digestion by proteinase K
diagnostics
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thermolysin is used in the diagnosis of prion diseases ovine scrapie and bovine spongiform encephalopathy, with similar sensitivity compared to proteinase K digestion, use of a protease to distinguish PrPC from PrPSc, overview
nutrition
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the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam
synthesis
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hydrolysis and condensation reactions of peptides catalyzed by enzyme can be reversibly controlled by on/off ultrasound irradiation depending on its frequency region
synthesis
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introduction of ionizing residues into the active site of enzyme as a means of modifying its pH-activity profile
synthesis
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immobilized enzyme catalyzes the formation of beta-cyclodextrin esters using vinyl esters of butyrate, decanoate and laurate, as acyl donors in dimethylsulfoxide. Esterification occurs exclusively at the glucose C-2 position. Enzyme also catalyzes the synthesis of alpha-, beta-, gamma- and maltosyl-beta-cyclodextrin esters with vinyl laurate as the acyl donor in dimethylsulfoxide and dimethylformamide
synthesis
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immobilized enzyme catalyzes the transesterification of vinyl laurate to several sucrose-containing tri- and tetrasaccharides. Preferred position of acylation is the 2-OH group of the alpha-D-glucopyranose moiety linked 1 to 2 to the beta-D-fructofuranose unit
synthesis
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the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam
synthesis
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thermolysin is industrially used for the synthesis of N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester, a precursor of an artificial sweetener aspartame, from N-carbobenzoxy-L-aspartic acid and L-phenylalanine methyl ester
industry
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the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam
synthesis
-
the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam
industry
Geobacillus stearothermophilus MK232
-
the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam
-
synthesis
Geobacillus stearothermophilus MK232
-
the enzyme is used for synthesis of N-carbobenzyloxy L-Asp-L-Phe methyl ester, a precursor of the artificial sweetener aspartam
-
medicine
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thermolysin degrades cellular prion protein while preserving both proteinase K-sensitive and proteinase K-resistant isoforms of disease-related prion protein in both rodent and human prion strains. In variant Creutzfeldt-Jakob disease, up to 90% of total prion protein present in the brain resists degradation with thermolysin, whereas only about 15% of this material resists digestion by proteinase K
additional information
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thermolysin treatment improves the yield of human intestinal epithelial cells. The thermolysin and endothelin-3 method can be used to isolate and generate viable human intestinal epithelial cells from the human small intestine
additional information
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thermolysin crystal can be used as the stationary phase in liquid chromatography to separate D/L-phenylglycine, thermolysin crystal is useful for chiral separation