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(dimethylamino)-1-naphthalenesulfonyl fluoride
-
100% inhibition of enzyme activity with N-trans cinnamoyl imidazole as substrate, during prolonged exposure to organic solvents the active-site fluorescent label inhibitor adopts a different binding conformation
1,10-phenanthroline
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 40%
2,2'-[[(1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphoryl]bis(oxybenzene-4,1-diyl)]diacetic acid
-
-
2,2'-[[(1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphoryl]bis(oxybenzene-4,1-diyl)]diacetic acid
-
-
2,2'-[[(1-[[1-(tert-butoxycarbonyl)-L-prolyl]amino]-3-methylbutyl)phosphoryl]bis(oxybenzene-3,1-diyl)]diacetic acid
-
-
2-phenylethaneboronic acid
-
-
2-phenylethanesulfonic acid
-
-
3,4-dichloroisocoumarin
-
-
4-(2-aminoethyl)-benzenesulfonyl fluoride
-
-
4-(2-aminoethyl)benzenesulfonyl fluoride
-
-
4-(4'-Aminophenylazo)phenylarsonic acid
-
-
4-bromophenacyl bromide
-
-
5-Dimethylaminonaphthalene-1-sulfonate
-
circular-dichroism and fluorescence studies of 5-dimethylaminonaphthalene-1-sulfonyl derivative of subtilisin indicate a closely similar structure to that of native subtilisin
acetonitril
high concentrations of acetonitrile significantly decrease the enzyme activity by over 60%, most likely due to disrupting the enzyme structural integrity
alpha2-Macroglobulin
-
inhibits alkaline proteolytic activity of purified Arp by 80%
-
angiotensin-converting enzyme
-
IC50 is 0.067 mg/ml
-
Benzyloxycarbonyl-(Ala)n-PheCH2Cl
-
benzyloxycarbonyl-(Ala)2-PheCH2Cl is the best inhibitor
Benzyloxycarbonyl-Ala-XaaCH2Cl
-
Xaa : Gly, Val, Ala, Leu, Phe
Benzyloxycarbonyl-L-phenylalanylbromomethane
-
reactivity is about an order of magnitude less than that of subtilisins BPN' and Carlsberg
bis(2,3,5-trimethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(2,3-dimethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(2,3-dimethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(2,5-dimethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(2-methylphenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(2-methylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(3,4,5-trimethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(3,4,5-trimethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(3,4-dimethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(3,4-dimethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(3-chlorophenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(3-chlorophenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(3-methoxyphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(4-chlorophenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(4-chlorophenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(4-ethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(4-ethylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(4-methoxyphenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(4-methylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis(4-tert-butylphenyl) (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis(4-tert-butylphenyl) (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis[4-(methylsulfonyl)phenyl] (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis[4-(propan-2-yl)phenyl] (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis[4-(propan-2-yl)phenyl] (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
bis[4-(sulfanylmethyl)phenyl] (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
bis[4-(sulfanylmethyl)phenyl] (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
Brij 35
at 37°C and pH of 7.5, 0.01% reduces prosubtilisin JB1 relative activity to 30% and 0.05% reduces prosubtilisin JB1 relative activity to 70%
Broad bean extract
-
subtilisin BPN'
Ca2+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 57% and 5 mM reduces prosubtilisin JB1 relative activity to 45%
Chiral amine- and aminoalcohol-derivatives
-
-
-
Chymotrypsin I inhibitor from potato
-
subtilisin Carlsberg, subtilisin BPN'
-
chymotrypsin inhibitor 2
-
-
-
chymotrypsin inhibitor 2 mutant M59A
-
-
-
chymotrypsin inhibitor 2 mutant M59F
-
-
-
chymotrypsin inhibitor 2 mutant M59G
-
-
-
chymotrypsin inhibitor 2 mutant M59K
-
-
-
chymotrypsin inhibitor 2 mutant M59Y
-
-
-
chymotrypsin inhibitor 2 mutant Y61A
-
-
-
Co2+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 14% and 5 mM reduces prosubtilisin JB1 relative activity to 24%
CrSPI-1
-
Kazal-type inhibitor from the hepatopancreas of the Carcinoscorpius rotundicauda, potently inhibits subtilisin
-
diisopropyl fluorophosphate
-
-
diisopropylphosphofluoridate
-
-
Dipeptidyl chloromethyl ketones
-
-
diphenyl (1-[[(benzyloxy)carbonyl]amino]-2-phenylethyl)phosphonate
-
-
diphenyl (1-[[(benzyloxy)carbonyl]amino]-3-methylbutyl)phosphonate
-
-
E-64c
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 25%
EGTA
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 34%
EPI1a
-
four-cysteine atypical Kazal-domain of protease inhibitor EPI1 from Phytophthora infestans. 80% inhibition at 0.00015 mM
-
EPI1b
-
typical Kazal-domain of protease inhibitor EPI1 from Phytophthora infestans, little inhibitory effect
-
fungal protease inhibitor F
-
specific inhibitor toward subtilisin-type protease. P1 residue most signficantly affects inhibitory specificity. Mutant T29M has stronger subtilisin-inhibitory activity than the wild-type, mutants T29E and T29R are relatively weaker inhibitors. Inhibitory activities of mutants T29F and T29L are as strong as that of the wild-type
-
guanidine hydrochloride
-
at 2 M inhibits subtilisin-like serine protease by 35% and at 4 M almost completely, whereas it has no inhibitory effect on subtilisin
guanidinium hydrochloride
-
H2O2
-
the enzyme activity is reduced to 68% after a 20-min incubation with 0.33% hydrogen peroxide at 25°C in Tris buffer, substrate suc-Ala-Ala-Pro-Phe-4-nitroanilide, recombinant enzyme
Hg+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 33% and 5 mM reduces prosubtilisin JB1 relative activity to 22%
human LEKTI
-
noncompetitive inhibition
-
human proteinase inhibitor 9
-
PI9, serine proteinase inhibitor
-
Inhibitor from Dolichos biflorus
-
purification and properties
-
Inhibitor from egg white
-
Inhibitor from seeds of Canavalia lineata
-
Inhibitor from seeds of Setaria italica
-
purification and characterization
-
Inhibitor from Streptomyces sp.
-
-
-
Inhibitor from Streptomyces virginiae
-
primary structure
-
Inhibitor from Vigna unguiculata subsp. cylindrica
-
Inhibitor of Amaranthus caudatus seeds
-
Inhibitor of trypsin from soybean
-
-
-
IvaP I9 domain
the N-terminal propeptide of enzyme IvaP, the IvaP I9 domain, can temporarily inhibit, and be cleaved by, the purified enzyme
-
K+
at 37°C and pH of 7.5, with 1 mM results in an almost complete reduction of prosubtilisin JB1 activity, 5 mM reduces prosubtilisin JB1 relative activity to 53%
L-[(1R)-1-acetamido-2-(1-naphthyl)ethyl]boronic acid
-
leupeptin
-
inhibits the enzymatic activity by 20%
Mg2+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 79% and 5 mM reduces prosubtilisin JB1 relative activity to 48%
N-((tert-Butoxycarbonyl)alanylprolylphenylalanyl)-O-benzoylhydroxylamine
-
-
N-(tert-butoxycarbonyl)-L-valyl-N-(1-[bis[4-(sulfanylmethyl)phenoxy]phosphoryl]-2-phenylethyl)-L-prolinamide
-
-
N-(tert-butoxycarbonyl)-L-valyl-N-[1-(diphenoxyphosphoryl)-2-phenylethyl]-L-prolinamide
-
-
N-(tert-butoxycarbonyl)-L-valyl-N-[1-[bis(3,4,5-trimethylphenoxy)phosphoryl]-3-methylbutyl]-L-prolinamide
-
-
N-(tert-butoxycarbonyl)-L-valyl-N-[1-[bis(4-methoxyphenoxy)phosphoryl]-3-methylbutyl]-L-prolinamide
-
-
N-(tert-butoxycarbonyl)-L-valyl-N-[1-[bis(4-tert-butylphenoxy)phosphoryl]-3-methylbutyl]-L-prolinamide
-
-
N-acetyl-L-tryptophan amide
molecular dynamics simulations are performed with subtilisin in the presence and in the absence of an inhibitor both in hexane and in water. The inhibitor induces an open conformation of the S1 pocket that is maintained after the removal of the ligand in anhydrous, but not in aqueous, simulations. The analysis of fluctuations suggest that this behavior is caused by the decreased flexibility exhibited by subtilisin in hexane
N-benzoyl-L-Arg
-
product inhibition
N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone
-
synthetic inhibitor
N-ethylmaleimide
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 15%
N-tosyl-L-phenylalanyl chloromethyl ketone
-
-
N1-18[ISP]
synthetic peptide corresponding to the N-terminal extension behaves as a mixed noncompetitive inhibitor of active ISP
neurolysin
-
IC50 is 0.263 mg/ml
-
p-chloromercuribenzoate
-
partial inhibition
p-Nitrophenylarsonate
-
-
pepstatin
-
almost completely inhibits acid proteolytic activity, does not inhibit alkaline proteolytic activity of purified Arp
pepstatin A
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 48%
Peptidyl chloromethyl ketones
-
-
phenylmethanesulfonyl fluoride
phenylmethylsulfonyl fluoride
Phenylmethylsulphonyl fluoride
-
-
Potato extract
-
subtilisin BPN'
-
propeptide
-
inhibition in a concentration-dependent manner. Wild-type propeptide is more potent than G56W-, G56S- and G56E-propeptide
-
Suc-Val-Pro-PheP(OPh)2
-
-
tert-butyl (2S)-2-([1-[bis(2-methylphenoxy)phosphoryl]-3-methylbutyl]carbamoyl)pyrrolidine-1-carboxylate
-
-
tert-butyl (2S)-2-([1-[bis(3,4,5-trimethylphenoxy)phosphoryl]-3-methylbutyl]carbamoyl)pyrrolidine-1-carboxylate
-
-
tert-butyl (2S)-2-([1-[bis(3,4-dimethylphenoxy)phosphoryl]-3-methylbutyl]carbamoyl)pyrrolidine-1-carboxylate
-
-
tert-butyl (2S)-2-([1-[bis(4-methoxyphenoxy)phosphoryl]-3-methylbutyl]carbamoyl)pyrrolidine-1-carboxylate
-
-
tert-butyl (2S)-2-([1-[bis(4-methylphenoxy)phosphoryl]-3-methylbutyl]carbamoyl)pyrrolidine-1-carboxylate
-
-
tert-butyl (2S)-2-[(1-[bis[4-(sulfanylmethyl)phenoxy]phosphoryl]-2-phenylethyl)carbamoyl]pyrrolidine-1-carboxylate
-
-
tert-butyl (2S)-2-[[1-(diphenoxyphosphoryl)-2-phenylethyl]carbamoyl]pyrrolidine-1-carboxylate
-
-
tomato inhibitor-II
-
TI-II, enzyme binding structure, the interdomain interface in TI-II consists of a small cluster of highly conserved hydrophobic residues Ile14, Pro16, Tyr98, Phe100 and Phe106 from domain I and Tyr34, Pro54 and Lys55 from domain II. Although this interface is quite small (buried surface area of 487A), it forms a stable packing arrangement between the two domains. Each reactive site loop in TI-II interacts with a separate molecule of subtilisin in the canonical manner observed in other proteinase–inhibitor complexes. The domains of TI-II appear to bind the proteinase independently of each other
-
tosyl-Phe chloromethyl ketone
Triton X-100
at 37°C and pH of 7.5, 0.01% reduces prosubtilisin JB1 relative activity to 92% and 0.05% reduces prosubtilisin JB1 relative activity to 69%
turkey egg white inhibitor
-
Tween 20
at 37°C and pH of 7.5, 0.01% reduces prosubtilisin JB1 relative activity to 53% and 0.05% reduces prosubtilisin JB1 relative activity to 70%
Urea
high concentrations (6 M) of urea significantly decrease the enzyme activity by over 60%, most likely due to disrupting the enzyme structural integrity
1,4-dioxane
-
104fold decrease in the enzymeÂ’s catalytic activity for the hydrolysis reaction of vinyl butyrate with D2O and a 50% decrease in enzyme structural dynamics. Attaching increasing amounts of PEG to the enzyme reverses some of the activity loss. Poly(ethylene)-glycolylation increases protein structural dynamics in 1,4-dioxane
1,4-dioxane
-
reduces the enzyme activity. Structural changes, flexibility, hydration, and changes of the enzyme ionization state are not responsible for the low storage stability. Possible depletion or rearrangement of water molecules around the active site, or small structural perturbations around the active site or movements of counter ions
antipain
-
-
antipain
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 75%
antipain
-
totally inhibits recombinant Sub2
antipain
35% inhibition at 1 mM, 95% at 5 mM
Aprotinin
-
-
Aprotinin
-
inhibits the enzymatic activity by 15%
chymostatin
-
-
chymostatin
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 95%
chymostatin
-
totally inhibits recombinant Sub2
chymostatin
complete inhibition at 1 mM
Cu2+
-
strain DJ-4, strong inhibition
Cu2+
at 37°C and pH of 7.5, with 1 mM results in an almost complete reduction of prosubtilisin JB1 activity, 5 mM reduces prosubtilisin JB1 relative activity to 19%
E-64
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 31%
E-64
37% inhibition at 5 mM
EDTA
inactivates the enzyme at 0.01 mM, the enzyme protein becomes less structured and potentially monomeric. Removal of Ca2+ at sites close to the dimer interface and the S1 pocket are involved enzyme inhhibition by EDTA
EDTA
5 mM partially inhibits by 35%, in 100 mM Tris-HCl buffer, pH 7.0 at 15°C for 20 min
EDTA
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 52%
EDTA
-
subtilisin exhibits little activity at 80°C in the presence of 10 mM. Subtilisin-like serine protease is fully active even in the presence of 10 mM EDTA
Inhibitor from egg white
-
subtilisin Carlsberg, subtilisin BPN'
-
Inhibitor from egg white
-
-
-
Inhibitor from seeds of Canavalia lineata
-
low MW protein of about 6500 MW
-
Inhibitor from seeds of Canavalia lineata
-
MW 22000, Kunitz type inhibitors
-
Inhibitor from Vigna unguiculata subsp. cylindrica
-
inactive with other enzymes
-
Inhibitor from Vigna unguiculata subsp. cylindrica
-
properties and kinetics of the inhibitor
-
Inhibitor of Amaranthus caudatus seeds
-
subtilisin Novo (BPN') from Bacillus amyloliquefaciens, subtilisin Carlsberg from Bacillus licheniformis
-
Inhibitor of Amaranthus caudatus seeds
-
subtilisin Novo (BPN') from Bacillus amyloliquefaciens, subtilisin Carlsberg from Bacillus licheniformis
-
nitrogen
-
together with carbon significantly reduces protease activity of KV01 at 16 and 36 h, and KV54 at 16 h
nitrogen
-
absence of C or N metabolite repression in subtilisins from KV42 isolate. Together with carbon significantly reduces protease activity of KV71 at 16 and 36 h, and KV22 at 16 h
phenylmethanesulfonyl fluoride
-
-
phenylmethanesulfonyl fluoride
-
-
phenylmethanesulfonyl fluoride
-
totally inhibits recombinant Sub2
phenylmethylsulfonyl fluoride
-
1 mM, 82% inhibition
phenylmethylsulfonyl fluoride
1 mM completely inhibits, in 100 mM Tris-HCl buffer, pH 7.0 at 15°C for 20 min
phenylmethylsulfonyl fluoride
-
-
phenylmethylsulfonyl fluoride
-
strain DJ-4
phenylmethylsulfonyl fluoride
-
-
phenylmethylsulfonyl fluoride
-
1 mM, 5% residual activity
phenylmethylsulfonyl fluoride
at 37°C and pH of 7.5, 0.1 mM inhibits prosubtilisin JB1 by 79%
phenylmethylsulfonyl fluoride
-
inhibits alkaline proteolytic activity of purified Arp by 100%, does not inhibit acid proteolytic activity
PMSF
-
-
PMSF
-
circular-dichroism and fluorescence studies of PMSF derivative of subtilisin indicate a closely similar structure to that of native subtilisin
PMSF
complete inhibition at 10 mM, no effect at 0.01 mM
Sodium dodecyl sulfate
-
activity of the enzyme is retarded by 2.3 and 244times in 1 mM and 40 mM sodium dodecyl sulfate respectively compared to that in buffer solution. No evidence of sandwich-like subtilisin–sodium dodecyl sulfate complex formation, thus the enzyme does not encroach into the hydrophobic surfactant core of sodium dodecyl sulfate micelle to form an elongated structure. Retains its structural integrity in sodium dodecyl sulfate solution. Micellar crowding in the vicinity of the enzyme
Sodium dodecyl sulfate
at 37°C and pH of 7.5, 0.01% reduces prosubtilisin JB1 relative activity to 52% and 0.05% reduces prosubtilisin JB1 relative activity to 29%
sucrose
-
together with carbon significantly reduces protease activity of KV01 at 16 and 36 h, and KV54 at 16 h
sucrose
-
absence of C or N metabolite repression in subtilisins from KV42 isolate. Together with carbon significantly reduces protease activity of KV71 at 16 and 36 h, and KV22 at 16 h
tosyl-Phe chloromethyl ketone
-
not
tosyl-Phe chloromethyl ketone
-
-
turkey egg white inhibitor
-
-
-
turkey egg white inhibitor
-
-
-
Zn2+
-
strain DJ-4, strong inhibition
Zn2+
-
1 mM, 59% residual activity
Zn2+
at 37°C and pH of 7.5, 1 mM reduces prosubtilisin JB1 relative activity to 30%, with 5 mM results in an almost complete reduction of prosubtilisin JB1 activity
additional information
-
not inhibited by 1,10 phenanthroline
-
additional information
-
not inhibitory: Tween-20, Tween-40, Tween-60, Tween-80 at 1%, sodium dodecylsulfate at 0.2%, for 1 h at 30°C. Not inhibitory: iodoacetate, ethylacetimide, phenylglyoxal, N-ethylmaleimide, N-bromosuccinimide
-
additional information
-
enzyme-specific, thermostable inhibitor from Physarum polycephalum with molecular mass of 32-33 kDa, 50% inhibition at 0.00014 mM
-
additional information
-
aldehyde and fluoromethyketone (FMK)-based inhibitors inhibit SAS-1 and -2 reversibly and only CMK-based inhibitors irreversibly inhibit the enzyme
-
additional information
-
molecular dynamics simulations, residue displacement correlations, and inhibitor design based on the enzyme inhibitor comlpex of TI-II and subtilisin, overview
-
additional information
-
not: tosyl-leucine chloromethyl ketone
-
additional information
-
metal chelating reagents; not: tosyl-leucine chloromethyl ketone; sulfhydryl reagents
-
additional information
-
strain DJ-4, no inhibition by EDTA or leupeptin
-
additional information
-
not inhibitory: EDTA at 5 mM
-
additional information
-
not inhibitory: sodium dodecylsulfate at 1 mM
-
additional information
at 37°C and pH of 7.5, 0.1 mM leupeptin does not inhibit prosubtilisin JB1
-
additional information
enzyme destabilization in the presence of EDTA is due to chelation of a tightly bound calcium ion by EDTA, rather than destabilizing effects caused by EDTA on the structure
-
additional information
-
is not inhibited by 1,10-phenanthroline, EDTA, E64, bestatin, phosphoramidon and pepstatin A
-
additional information
-
not inhibited by 1,10 phenanthroline
-
additional information
-
alkaline proteolytic activity of purified Arp not inhibited by 3,4-dichloroisocumarin, tosyl lysine chloromethyl ketone, tosyl phenylalanine chloromethyl ketone, leupeptin, chymostatin, elastatinal, iodoacetamide, E-64, EDTA, 1,10-phenanthroline, aprotinin, antithrombin III, alpha1-antitrypsin and soybean trypsin inhibitor
-
additional information
the enzyme activity is not affected by various metal ions or non-specific protease inhibitors, e.g. bestatin, leupeptin, or pefabloc SC
-
additional information
-
enzymatic activity of mature subtilisin is inhibited by G56S-propeptide and wild-type propeptide in a concentration-dependent manner
-
additional information
-
subtilisin-like serine protease is fully resistant to treatment with 2-5% SDS, 4-8 M urea, 10% Tween-20 or 10% Triton X-100. In gel assay, subtilisin-like serine protease is fully denatured prior to SDS-PAGE by trichloroacetic acid treatment, followed by boiling for 5 min in the presence of SDS
-
additional information
no inhibition by EDTA and benzamidine
-