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PubMed1987

Methionine residue accessibility in native subtilisin DY.

The three methionine residues of subtilisin DY were specifically modified into methionine sulfoxide using increasing amounts of chloramine T. By means of subsequent treatment with cyanogen bromide, gel chromatography, Edman degradation of the obtained peptides and the known structure of subtilisin DY it was established that Met222 is exposed to the surrounding solution, Met124 is partially exposed and Met199 is buried. The data obtained were confirmed on a computer graphics space movable model of subtilisin Carlsberg where Met222 was seen to be on the surface of the molecule and Met199 shielded by Tyr262, Ala179 and Leu196. Upon oxidation of Met222 of subtilisin DY by chloramine T, 25% of its caseinolytic activity was lost. This can be explained by the immediate adjacency to the active-site Ser221. An additional 5% loss of activity was observed at each subsequent methionine modification.

Amino Acid Sequence↗
PubMed1987

Reductive alkylation of lysine residues in subtilisin DY.

Only lysine epsilon-amino groups (and the N-terminal alpha-amino group) in native subtilisin DY were reductively alkylated by glyceraldehyde in the presence of sodium cyanoborohydride. The modified protein molecule was cleaved by TosPheCH2Cl-trypsin or cyanogen bromide and the two sets of peptides obtained were fractionated and purified by gel filtration and HPLC. For determination of the degree of modification of each lysine residue, selected peptides were subjected to sequence analysis combined with quantitative estimation of the containing PTH-Lys and PTH-epsilon-DHP-Lys. The data obtained showed that the lysine residues in positions 12, 15, 27, 43, 136, 141, 265 were entirely modified, those in positions 170, 184, 237 were partially modified, and Lys22 and Lys94 were unaccessible for the reagent. The caseinolytic activity decreased by 23% when the maximum number of lysine residues (8.6 of the total 12 residues) in subtilisin DY were modified. The CD-spectra of native and modified enzyme showed only slight differences. Both these experiments suggest that the lysine residues do not take part directly in the catalytic reaction but are responsible for maintaining the native three-dimensional enzyme structure. The data obtained for the accessibility of the different lysine residues in subtilisin DY correlated very well with the positions of these residues in a video model of the structure of subtilisin Carlsberg, thus suggesting that the spatial structures of these two enzymes are very similar.

Alkylation↗
PubMed1978

[Immunochemical study of subtilisins obtained from Bacillus subtilis A-50 and some of its mutants].

Physico-chemical parameters of subtilisins from the original Bacillus subtilis A-50 strain (proteolytic activity, electrophoretic mobility, molecular weight, reactions with specific inhibitors) were similar to those mentioned in the literature for the enzymes of other strains. Immunological experiment has shown, that Bacillus subtilis A-50 subtilisins with various electrophoretic mobility do not differ in their antigenic properties. Enzymes with high electrophoretic mobility from mutant strains were similar to I--III subtilisin fractions from Bac. subtilis A-50 in the antigenic characteristics. However, the antigenic heterogeneity was observed in I, II and III enzyme fractions of some mutant strains. Subtilisins studied appear to form the isoenzyme system.

Antigen-Antibody Reactions↗
PubMed1999

[Peptide synthesis catalyzed by subtilisin-72 in organic solvents].

The solubility, stability, and activity of native subtilisin 72 and of its complex with SDS were comparatively studied in a number of polar organic solvents. Subtilisin was found to catalyze peptide bond formation when suspended in acetonitrile or solubilized as a complex with SDS in ethanol and isopropanol. Tripeptide Z-Ala-Ala-Leu-pNA, tetrapeptides A-Ala-Ala-P1-P1'-B, where A = Z or Abz; P1 = Leu, Phe, Met, Trp, Ile, Tyr, Phe(NO2), or Glu(OMe), P1' = Leu, Phe, Glu, Ala, Ile, Val, or Arg; B = NH2, pNA, or 2-(2,4-dinitrophenyl)aminoethylamine residue (Ded); pentapeptides Z-Ala-Ala-Leu-Ala-Ala-pNA and Z-Ala-Ala-Leu-Ala-Phe-pNA; and hexapeptide Abz-Val-Ala-Phe-Phe-Ala-Ala-Ded were synthesized using the SDS-subtilisin complex. The complex also efficiently catalyzed the oligomerization of tripeptide H-Phe-Ala-Leu-OCH3 in ethanol, which resulted in a 63:37 mixture of trioligomer and tetraoligomer. It was demonstrated that SDS-subtilisin is a much more efficient catalyst than the suspension of native enzyme.

Catalysis↗
PubMed1979

[Affinity chromatography of subtilisin on a sorbent with epsilon-aminocapronyl-alanyl-alanyl-D-leucylamide. Detection of a serine protease with unusual properties].

A biospecific sorbent obtained by attachment of epsilon-aminocapronyl-L-alanyl-L-alanyl-D-leucylamide to CNBr-activated Sepharose 4B has been used for affinity chromatography of various samples of subtilisin BPN', e.g. subtilisin A ("Serva"), Nagarse, A-50. Two active components were isolated from subtilisin A (Serva"), the major component corresponding to subtilisin BPN' and the minor component (SII) being a serine proteinase with low molecular weight (about 10000). The molecular weight and amino acid composition of SII as well as the kinetic parameters of its action on peptide substrates (p-nitroanilides of N-benzyloxycarbonyl-Gly-Gly-Leu, -Ala-Ala-Leu, -Gly-Gly-Phe, -Ala-Ala-Phe. The low molecular weight proteinase possesses a high affinity for the leucine residue in P1 position and alanine in P2 and P3 positions. The specificity of this proteinase differs from that of the main component.

Chromatography, Affinity↗
PubMed1994

The sequence of a subtilisin-type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability.

The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 degrees C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80-130 degrees C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp. The total DNA sequence of the gene encoding the protease (named "aerolysin") was determined. Multiple sequence alignment with 15 different serine-type proteases showed greatest homology with subtilisins from gram-positive bacteria rather than archaeal or eukaryal serine proteases. Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilum subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha-helices. Aspartic acids were found at the N-terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.

Amino Acid Sequence↗
PubMed1997

Isolation of a cDNA encoding a novel subtilisin-like protease (Pr1B) from the entomopathogenic fungus, Metarhizium anisopliae using differential display-RT-PCR.

Reverse transcription differential display PCR (RT-DD-PCR) was used to identify genes that are specifically expressed by Metarhizium anisopliae when it contacts the host insect cuticle. Using a homology-based subtilisin-like protease primer we identified a hitherto unsuspected differentially expressed subtilisin-like protease (Pr1B) encoding gene. The deduced amino acid sequence shows 54% similarity to the well characterized Pr1A subtilisin of M. anisopliae and karyotype analysis revealed that Pr1A and Pr1B are located on separate chromosomes. Like Pr1A, Pr1B is synthesized as a large precursor (1158 nucleotides; deduced molecular mass = 40031 Da) containing a signal peptide, a propeptide and the mature protease (283 aa; deduced molecular mass = 28714 Da). However, Pr1B possesses several substitutions in the highly conserved sequences comprising the active sites of subtilisins. In particular, the substitution of Thr220 by serine is unique to Pr1B. Substitution of Asn155 by glycine is also very unusual, and we discuss the likely effects these changes will have on the catalytic efficiency of Pr1B.

Amino Acid Sequence↗
PubMed1987

Protein engineering of subtilisin BPN': enhanced stabilization through the introduction of two cysteines to form a disulfide bond.

Introduction of a disulfide bond by site-directed mutagenesis was found to enhance the stability of subtilisin BPN' (EC 3.4.21.14) under a variety of conditions. The location of the new disulfide bond was selected with the aid of a computer program, which scored various sites according to the amount of distortion that an introduced disulfide linkage would create in a 1.3-A X-ray model of native subtilisin BPN'. Of the several amino acid pairs identified by this program as suitable candidates, Thr-22 and Ser-87 were selected by using the additional requirement that the individual cysteine substitutions occur at positions that exhibit some degree of variability in related subtilisin amino acid sequences. A subtilisin variant containing cysteine residues at positions 22 and 87 was created by site-directed mutagenesis and was shown to have an activity essentially equivalent to that of the wild-type enzyme. Differential scanning calorimetry experiments demonstrated the variant protein to have a melting temperature 3.1 degrees C higher than that of the wild-type protein and 5.8 degrees C higher than that of the reduced form (-SH HS-) of the variant protein. Kinetic experiments performed under a variety of conditions, including 8 M urea, showed that the Cys-22/Cys-87 disulfide variant undergoes thermal inactivation at half the rate of that of the wild-type enzyme. The increased thermal stability of this disulfide variant is consistent with a decrease in entropy for the unfolded state relative to the unfolded state that contains no cross-link, as would be predicted from the statistical thermodynamics of polymers.

Anti-Bacterial Agents↗
PubMed2003

Expression of subtilisin-like serine proteases in Arabidopsis thaliana is cell-specific and responds to jasmonic acid and heavy metals with developmental differences.

The expression of two novel subtilisin-like serine proteases At-SLP2 and At-SLP3 from Arabidopsis thaliana and the recently identified Arabidopsis subtilase ARA12 was analysed with respect to plant development, stress response and cell specificity. In juvenile plants the mRNAs of the subtilisin-like proteases At-SLP2, At-SLP3 and ARA12 were detected with varying transcript levels in leaves but not in roots. In mature Arabidopsis plants transcripts were abundant in leaves, roots and flowers revealing developmental regulation of synthesis of subtilases. By in situ hybridization it was shown that the subtilisin-like proteases were predominantly present in epidermal cells and in the vascular bundles, in the phloem and in developing xylem elements. In flowers additional signals were localized, for example, in pistils, ovules and anthers. In flowers and juvenile developing leaves, expression of the subtilisin-like proteases increased following treatment with jasmonate and cadmium, respectively, suggesting that these proteases are responsive to stress and pathogen stimuli. The physiological relevance of these data in relation to plant morphogenesis and development is discussed.

Journal Article↗
PubMed1972

Comparative studies of the specificities of -chymotrypsin and subtilisin BPN'. Studies with flexible substrates.

A series of arylalkanoate esters and alpha-acetamidoarylalkanoate esters were tested as substrates for alpha-chymotrypsin and subtilisin BPN'. Chymotrypsin hydrolysed N-acetyl-l-phenylalanine methyl ester and methyl 4-phenylbutyrate faster than their respective higher and lower homologues, whereas methyl 2-acetamido-6-phenylhexanoate and methyl 6-phenylhexanoate were better substrates for subtilisin than their lower homologues. N-Acetyl-l-tryptophan methyl ester and its analogue, N-acetyl-3-(1-naphthyl)-alanine methyl ester, were hydrolysed 23 times faster by chymotrypsin than by subtilisin. These results indicate that the binding site of alpha-chymotrypsin is roughly 1.1nm (11A) long and curved, whereas that of subtilisin is a longer system and less curved. The stereo-specificity during the hydrolysis of typical substrates by both enzymes was found to vary over a wide range. The enhancing effect of the alpha-acetamido group in the l-series of substrates and the detrimental effect in the d-series of substrates also varies considerably.

Binding Sites↗
PubMed1972

Comparative studies of the specificities of -chymotrypsin and subtilisin BPN'. Studies with flexible and 'locked' substrates.

Subtilisin BPN' hydrolysed N-acetyl-l-3-(2-naphthyl)-alanine methyl ester, N-acetyl-l-leucine methyl ester and N-acetyl-l-valine methyl ester, faster than alpha-chymotrypsin. Of eight ;locked' substrates tested, only methyl 5,6-benzindan-2-carboxylate was hydrolysed faster by subtilisin, whereas the other esters were better substrates for chymotrypsin. Compared with the values for chymotrypsin, the stereospecific ratios during the hydrolysis of the optically active locked substrates by subtilisin were decreased by one and two orders of magnitude for bi- and tri-cyclic substrates respectively. The polar groups adjacent to the alpha-carbon atom of locked substrates did not contribute significantly to the reactivity of the more active optical isomers, but had a detrimental effect on the less active antipodes during hydrolysis by both the enzymes. These studies show that the binding site of subtilisin BPN' is longer and broader than that of alpha-chymotrypsin.

Binding Sites↗
PubMed1993

Engineering of the substrate-binding region of the subtilisin-like, cell-envelope proteinase of Lactococcus lactis.

The substrate-binding region of the cell-envelope proteinase of Lactococcus lactis strain SK11 was modelled, based on sequence homology of the catalytic domain with the serine proteinases subtilisin and thermitase. Substitutions, deletions and insertions were introduced, by site-directed and cassette mutagenesis of the prtP gene encoding this enzyme, based on sequence comparison both with subtilisin and with the homologous L.lactis strain Wg2 proteinase, which has different proteolytic properties. The engineered enzymes were investigated for thermal stability, proteolytic activity and cleavage specificity towards small chromogenic peptide substrates and the peptide alpha s1-casein(1-23). Mutations in the subtilisin-like substrate-binding region showed that Ser433 is the active site residue, and that residues 138 and 166 at either side of the binding cleft play an important role in substrate specificity, particularly when these residues and the substrate are oppositely charged. The K748T mutation in a different domain also affected specificity and stability, suggesting that this residue is in close proximity to the subtilisin-like domain and may form part of the substrate-binding site. Several mutant SK11 proteinases have novel properties not previously encountered in natural variants. Replacements of residues 137-139AKT along one side of the binding cleft produced the 137-139GPP mutant proteinase with reduced activity and narrowed specificity, and the 137-139GLA mutant with increased activity and broader specificity. Furthermore, the 137-139GDT mutant had a specificity towards alpha s1-casein(1-23) closely resembling that of L.lactis Wg2 proteinase. Mutants with an additional negative charge in the binding region were more stable towards autoproteolysis.

Amino Acid Sequence↗
PubMed1999

A new subtilisin-like proteinase from roots of the dandelion Taraxacum officinale Webb S. L.

A serine proteinase from roots of Taraxacum officinale Webb S. L. was isolated by affinity chromatography and gel-filtration on Superose 6R using FPLC. The enzyme is a 67-kD glycoprotein containing 54% carbohydrate which we have named taraxalisin. The substrate specificity of taraxalisin toward synthetic peptides and oxidized insulin B-chain is comparable with that of cucumisin from Cucumis melo and the subtilisin-like serine proteinase macluralisin from Maclura pomifera. The proteinase is inactivated by DFP and PMSF. Taraxalisin exhibits maximal activity at pH 8.0. The pH range for stability of the enzyme is narrow--6.0-9.0. The temperature optimum for the subtilisin-like activity is 40 degrees C. The N-terminal sequence of taraxalisin has 40% of its residues identical to those of subtilisin Carlsberg. Thus, the serine proteinase from dandelion roots is a member of the subtilisin family, which is evidently widespread in the plant kingdom.

Amino Acid Sequence↗
PubMed2005

Kinetic and molecular properties of Bacillus subtilis IBTC-3 subtilisin.

Bacillus subtilis IBTC-3 subtilisin was purified by gel filtration on Sephadex G 75 and affinity chromatography on bacitracin-CNBr-Sepharose 4B and characterized. Its molecular mass of 27 kDa was determined by SDS-PAGE, and isoelectric pH of 8.4 by chromatofocusing. FT-Raman and FT-IR spectroscopy studies revealed fragments with alpha-helix and irregular secondary structures within the polypeptide chain. The beta-sheet conformation was observed only in second-derivatives of FT-RS and FT-IR spectra, in the range of the amide II, III, and I bands. Tyr residues were shown to be hydrogen bonded and CSCH(3) groups adopted two conformations (P(H)-T and P(C)-G conformers). Kinetic properties of B. subtilis IBTC-3 subtilisin in hydrolysis of ethyl esters of amino acid derivatives were compared with that of alkaline peptidase from Bacillus alcalophilus PB92. The first enzyme displayed the highest affinity for NAc-Phe-OEt, both in hydrolysis (K(m) of 0.22 mM) and in synthesis (K(m) of 0.85 mM), whereas PB92 peptidase preferred Tyr derivatives (NAc-Tyr-OEt, K(m) of 0.043 and 0.75 mM, respectively). In contrast to the latter enzyme, B. subtilis IBTC-3 subtilisin catalyzed hydrolysis and synthesis of Bz-Arg-OEt.

Bacillus subtilis↗
PubMed2002

Mycosin-1, a subtilisin-like serine protease of Mycobacterium tuberculosis, is cell wall-associated and expressed during infection of macrophages.

BACKGROUND: Exported proteases are commonly associated with virulence in bacterial pathogens, yet there is a paucity of information regarding their role in Mycobacterium tuberculosis. There are five genes (mycP1-5) present within the genome of Mycobacterium tuberculosis H37Rv that encode a family of secreted, subtilisin-like serine proteases (the mycosins). The gene mycP1 (encoding mycosin-1) was found to be situated 3700 bp (four ORF's) from the RD1 deletion region in the genome of the attenuated vaccine strain M. bovis BCG (bacille de Calmette et Guérin) and was selected for further analyses due to the absence of expression in this organism. RESULTS: Full-length, 50 kDa mycosin-1 was observed in M. tuberculosis cellular lysates, whereas lower-molecular-weight species were detected in culture filtrates. A similar lower-molecular-weight species was also observed during growth in macrophages. Mycosin-1 was localized to the membrane and cell wall fractions in M. tuberculosis by Western blotting, and to the cell envelope by electron microscopy. Furthermore, M. tuberculosis culture filtrates were shown to contain a proteolytic activity inhibited by mixed serine/cysteine protease inhibitors and activated by Ca2+, features typical of the subtilisins. CONCLUSIONS: Mycosin-1 is an extracellular protein that is membrane- and cell wall-associated, and is shed into the culture supernatant. The protein is expressed after infection of macrophages and is subjected to proteolytic processing. Although proteolytically active mycosin-1 could not be generated recombinantly, serine protease activity containing features typical of the subtilisins was detected in M. tuberculosis culture filtrates.

Animals↗
PubMed2003

Comparison of theoretical and experimental data to evaluate substrate diffusional limitations for crown ether- and methyl-beta-cyclodextrin-activated serine protease subtilisin Carlsberg in tetrahydrofuran.

Simple co-lyophilization of serine protease subtilisin Carlsberg with [12]-crown ether-4 (12-crown-4) or methyl-beta-cyclodextrin (MbetaCD) drastically increases its catalytic activity in organic solvents. We investigated whether the improved activity would cause substrate diffusional limitations. To experimentally assess the issue, the enzyme was inactivated with PMSF. Different amounts of active and inactive subtilisin were codissolved in 10 mM phosphate buffer (pH 7.8) followed by lyophilization with or without 12-crown-4 or MbetaCD. Initial rates for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester and 1-propanol in anhydrous THF were plotted vs. the amount of active enzyme present in the formulations. For all three enzyme formulations a linear relationship was observed and the results clearly show that activation of subtilisin Carlsberg by crown ethers and MbetaCD did not cause diffusional limitations. This was somewhat surprising because theoretical models predicted such diffusional limitations for the activated formulations. However, investigation of the protein powder particles obtained after co-lyophilization with 12-crown-4 and MbetaCD revealed a drastically reduced particle size for these formulations when suspended in THF. The particle micronization afforded by the excipients prevented substrate diffusional limitations, a factor that should be taken into account when designing improved enzyme formulations for synthetic applications in organic solvents.

Coenzymes↗
PubMed1998

Stabilizing the subtilisin BPN' pro-domain by phage display selection: how restrictive is the amino acid code for maximum protein stability?

We have devised a procedure using monovalent phage display to select for stable mutants in the pro-domain of the serine protease, subtilisin BPN'. In complex with subtilisin, the pro-domain assumes a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated, however, the pro-domain is 97% unfolded. These experiments use combinatorial mutagenesis to select for stabilizing amino acid combinations at a particular structural locus and determine how many combinations are close to the maximum protein stability. The selection for stability is based on the fact that the independent stability of the pro-domain is very low and that binding to subtilisin is thermodynamically linked to folding. Two libraries of mutant pro-domains were constructed and analyzed to determine how many combinations of amino acids at a particular structural locus result in the maximum stability. A library comprises all combinations of four amino acids at a structural locus. Previous studies using combinatorial genetics have shown that many different combinations of amino acids can be accommodated in a selected locus without destroying function. The present results indicate that the number of sequence combinations at a structural locus, which are close to the maximum stability, is small. The most striking example is a selection at an interior locus of the pro-domain. After two rounds of phagemid selection, one amino acid combination is found in 40% of sequenced mutants. The most frequently selected mutant has a deltaG(unfolding) = 4 kcal/mol at 25 degrees C, an increase of 6 kcal/mol relative to the naturally occurring sequence. Some implications of these results on the amount of sequence information needed to specify a unique tertiary fold are discussed. Apart from possible implications on the folding code, the phage display selection described here should be useful in optimizing the stability of other proteins, which can be displayed on the phage surface.

Amino Acid Sequence↗
PubMed1997

Human proteinase inhibitor 9 (PI9) is a potent inhibitor of subtilisin A.

Serine proteinase inhibitors function as regulators of serine proteinase activity in a variety of physiological processes. Proteinase inhibitor 9 (PI9) is a 42 kDa member of the ovalbumin family of serpins that is expressed in placenta, lung, and cytotoxic lymphocytes. In this study, we have described the inhibitory mechanism of recombinant human PI9 towards the bacterial endoproteinase subtilisin A. PI9 inhibited the amidolytic activity of subtilisin A via a rapid, single step mechanism with an equilibrium inhibition constant of 3.6 pM and an overall second-order association rate constant of 2.4 x 10(6) M-1s-1, which is the strongest inhibitory mechanism of PI9 that has been described. The inhibitory action of PI9 towards subtilisin as a model proteinase may yield some indication of potential proteinases that may be regulated by PI9 in vivo.

Enzyme Inhibitors↗