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PubMed1992

Subtilisin from Bacillus subtilis strain 72. The influence of substrate structure, temperature and pH on catalytic properties.

Kinetic constants for the hydrolysis of the series of p-nitroanilide peptide substrates catalyzed by subtilisin from Bacillus subtilis strain 72 have been determined. The series of N-protected p-nitroanilides of the Z-A2-A1-pNA, Z-A3-A2-A1-pNA, Z-A4-A3-A2-A1-pNA types (Z-, benzyloxycarbonyl-1; -pNA, p-nitroanilide; A1-An, amino acid residues of the L-configuration) have been used. Subsite S1 reveals a preference for hydrophobic amino acid residues, i.e., leucine and phenylalanine. A preference for Leu over Phe at this position is manifested at the catalytic step, but not during the binding process. The beta-branched (Val, Ile) and the basic (Arg) amino acid residues cannot interact with the S1 subsite and the hydrolysis of the corresponding peptides occurs exclusively at the A2-A1 bond. If S1/A1 interactions are weak (Ala, Nva, Nle), the amino acid residue A1 can interact with subsites S1 and S'1 resulting in the hydrolysis at two bonds (A1-pNA and A2-A1). The data obtained suggests that the S'1 subsite is of broad selectivity. Subsite S2 reveals a preference for small amino acid residues. At pH 5.5-9 and below 50 degrees C, the subtilisin study does not lose its activity. At higher temperatures a rapid thermoinactivation occurs. Substrate binding stabilizes the enzyme. The temperature dependences of the kinetic and thermodynamic parameters suggest that the enzyme exists in two, i.e., 'cold' and 'hot' forms. At 22 degrees C the 'cold' form turns into the 'hot' one possibly owing to a conformational change. The enzyme-substrate complex does not exhibit such behavior and exists in only one form in the whole temperature range studied. The activity of an uncomplexed enzyme is controlled by a group of pKa = 7.2 +/- 0.1, which probably belongs to the histidine imidazole.

Amino Acid Sequence↗
PubMed1993

Identification of autoproteolytic cleavage site in the Asp-49 mutant subtilisin J by site-directed mutagenesis.

Ser-49 located at the exposed surface loop of subtilisin J was replaced with Asp and Arg. Proteinase activity of the Asp-49 mutant was similar to that of wild-type, but the Arg mutant was inactivated. At 37 degrees C, mature subtilisin J protein of the Asp-49 mutant rapidly degraded, and specific breakdown products were accumulated. These proteins were analyzed by SDS-PAGE, and the N-terminal sequences were determined for the mature and deleted protein. We identified the autoproteolytic cleavage site in the mature Asp-49 mutant protein from sequencing data.

Amino Acid Sequence↗
PubMed1996

New subtilisin-trypsin inhibitors produced by Streptomyces: primary structures and their relationship to other proteinase inhibitors from Streptomyces.

Three new proteinaceous inhibitors of trypsin and subtilisin of the Streptomyces subtilisin inhibitor (SSI)-like (SIL) protein family were isolated and purified from culture media of Streptomyces strains; SIL5 from S. fradiae, SIL7 from S. ambofaciens and SIL12 from S. hygroscopicus. Their complete amino-acid sequences were determined by sequence analysis of the intact SIL proteins and peptides obtained by enzymatic digestion of S-pyridylethylated proteins. SIL7 showed high sequence similarity to other Arg-possessing SSI-family inhibitors at the P1 site. SIL12 is unique in having a two-residue insertion in the flexible loop region. Based on the amino-acid sequences of these inhibitors and other SSI-family inhibitors whose sequences have already been determined, the phylogenetic relationship of SSI-family inhibitors and Streptomyces strains was considered. Among about 110 amino-acid residues possessed by SSI-family inhibitors, 28 are completely conserved. The contribution of these conserved residues to the function and stability of the inhibitor molecules is discussed on the basis of the results obtained from mutational analysis of SSI and its crystal structure.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of the pepD gene of Aspergillus niger which codes for a subtilisin-like protease.

Serine proteases constitute an important group of extra- and intracellular proteases in fungi. These enzymes are characterized by conserved regions around the active site residues, Asp, His and Ser. Based on this amino acid (aa) sequence conservation, we have used degenerate primer PCR to isolate subtilisin-specific genomic probes from Aspergillus niger, and cloned a gene, pepD, by screening a lambda genomic library using a PCR probe. The pepD gene contains three putative introns, which are 51-, 47- and 55-bp long and has an open reading frame coding for a protein which consists of 416 aa. The deduced aa sequence shows similarity to subtilisin-like proteases, in particular to fungal alkaline proteases. Signal sequence cleavage prediction indicates that the first 20 aa are probably removed upon transfer to the endoplasmic reticulum. The conservation of the pro-enzyme cleavage site in fungal alkaline proteases suggests that the mature protein is derived from this polypeptide via the removal of an additional 101 aa, resulting in a mature 30,294-Da enzyme consisting of 295 aa.

Amino Acid Sequence↗
PubMed1995

Subtilisin and alpha-chymotrypsin catalyzed synthesis of peptides containing arginine and lysine p-nitroanilides as C-terminal moieties.

p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal arginine or lysine residues have been obtained, as a rule with good yields, via acylation of arginine or lysine p-nitroanilides by methyl esters of respective N-acylated peptides, catalyzed by subtilisin or alpha-chymotrypsin. The synthesis might be performed by two routes--by reaction in water-organic solvent mixtures, catalyzed by dissolved enzyme, or by condensation of the components in organic solvents with low water content in the presence of any enzyme distributed over a silica support surface. The second approach seems to be preferable due to suppression of hydrolytic side reactions and improved stability of an enzyme. Subtilisin 72 is especially effective as a catalyst for the acylation of p-nitroanilides by N-protected tripeptide methyl esters--the derivatives capable of occupying the S1, S2 and S3 subsites of its extended binding site. Even dipeptide esters with D-amino acids in P2 position can be applied for p-nitroanilide acylation. The efficiency of alpha-chymotrypsin as a catalyst for peptide synthesis is more limited due to restricted specificity of this enzyme.

Amino Acid Sequence↗
PubMed1988

Comparative characterization of the substrate-binding subsites in subtilisins DY and Carlsberg by fluorescence and kinetic studies.

Peptide chloromethanes with the general formula dansyl-(Ala)n-Phe-CH2Cl where n = 0, 1, 2, 3 and dansyl fluoride were used to investigate the substrate-binding sites A and B in subtilisins DY and Carlsberg. Kinetic evidence for the introduction of the dansyl group at the subsites S2, S3, S4 and S5 were obtained. Fluorescence experiments showed that the micro-environment of these subsites is quite apolar. However, some differences in their accessibility to external reagents can be revealed in fluorescence quenching experiments. Efficient singlet-singlet radiationless energy transfer from the single Trp 113 to the dansyl group selectively bound at the respective subsites was observed and intramolecular distances between the chromophores were determined. The values calculated for the pairs Trp 113 plus Dns at S2, Trp 113 plus Dns at S4 and Trp 113 plus Dns at S5 are practically identical (1.7-2.0 nm) for the two enzymes. Conclusions on the shape of the substrate-binding sites in subtilisins DY and Carlsberg are drawn. The mutual spatial orientation of the donor (Trp 113) and acceptor (Dns at Sn) dipoles is also elucidated.

Binding Sites↗
PubMed2003

Nucleotide and deduced amino acid sequences of a high-molecular-mass subtilisin from an alkaliphilic Bacillus isolate.

A high-molecular-mass subtilisin was found in culture broth of the alkaliphilic Bacillus sp. strain KSM-KP43. The gene encoding the enzyme (FT protease) was determined using a mixed primer designed from the N-terminal amino acid (aa) sequence of the purified enzyme. The determined nucleotide sequence of the gene consisted of a 2427-bp open reading frame (ORF) that encoded a putative prepro-peptide (152 aa) and a mature enzyme (656 aa; 68,506 Da). The deduced aa of the mature enzyme revealed a moderate homology to a subtilisin-type proteinase from Bacillus halodurans and a minor extracellular protease, Vpr, from Bacillus subtilis with 64% and 57% identity, respectively. The molecular mass of the purified recombinant FT protease was approximately 72 kDa as judged by both SDS-polyacrylamide gel electrophoresis (PAGE) and gel filtration. FT protease showed maximal activity toward glutaryl-Ala-Ala-Pro-Leu-p-nitroanilide at pH 10.5 and at 45 degrees C. The enzyme was rapidly inactivated by incubation over 45 degrees C for 15 min at both pH 7 and 10. Calcium ions were slightly protective for thermoinactivation of the enzyme.

Amino Acid Sequence↗
PubMed2005

Folding pathway mediated by an intramolecular chaperone: intrinsically unstructured propeptide modulates stochastic activation of subtilisin.

Several secreted proteases are synthesized with N-terminal propeptides that function as intramolecular chaperones (IMCs) and direct the folding of proteases to their native functional states. Using subtilisin E as our model system, we had earlier established that (i) release and degradation of the IMC from its complex with the protease upon completion of folding is the rate-determining step to protease maturation and, (ii) IMC of SbtE is an extremely charged, intrinsically unstructured polypeptide that adopts an alpha-beta structure only in the presence of the protease. Here, we explore the mechanism of IMC release and the intricate relationship between IMC structure and protease activation. We establish that the release of the first IMC from its protease domain is a non-deterministic event that subsequently triggers an activation cascade through trans-proteolysis. By in silico simulation of the protease maturation pathway through application of stochastic algorithms, we further analyze the sub-stages of the release step. Our work shows that modulating the structure of the IMC domain through external solvent conditions can vary both the time and randomness of protease activation. This behavior of the protease can be correlated to varying the release-rebinding equilibrium of IMC, through simulation. Thus, a delicate balance underlies IMC structure, release, and protease activation. Proteases are ubiquitous enzymes crucial for fundamental cellular processes and require deterministic activation mechanisms. Our work on SbtE establishes that through selection of an intrinsically unstructured IMC domain, nature appears to have selected for a viable deterministic handle that controls a fundamentally random event. While this outlines an important mechanism for regulation of protease activation, it also provides a unique approach to maintain industrially viable subtilisins in extremely stable states that can be activated at will.

Algorithms↗
PubMed1988

Yeast KEX2 genes encodes an endopeptidase homologous to subtilisin-like serine proteases.

Yeast Saccharomyces cerevisiae KEX2 gene previously isolated, was characterized as the gene encoding a calcium-dependent endopeptidase required for processing of precursors of alpha-factor and killer toxin. In this study, we report the amino acid sequence of the KEX2 gene product deduced from nucleotide sequencing. Our results indicate that the KEX2 gene contains a 2,442-bp open reading frame encoding a polypeptide of 814 amino acids. The deduced amino acid sequence contains a region extensively homologous to the members of subtilisin-like serine protease family near the N-terminus. A putative membrane-spanning domain near the C-terminus was also detected. These facts indicate that the KEX2-encoded protein may function as a membrane-bound, subtilisin-like serine protease.

Amino Acid Sequence↗
PubMed1998

Subtilisin-catalyzed esterification of di- and oligosaccharides containing a D-fructose moiety.

Several di- and oligosaccharides containing a D-fructose moiety have been acylated by protease subtilisin in anhydrous dimethylformamide in the presence of the activated ester trifluoroethyl butanoate. Under the reaction conditions used, all the substrates were converted into the corresponding monobutanoates in ca. 50% isolated yields. Structural determination of the products by 13C NMR indicated a strong preference of subtilisin towards the regioselective esterification of the primary hydroxyls of the fructose moiety and, specifically, of the C-1 OH, as already observed with sucrose.

Acylation↗
PubMed1990

Microenvironmental contributions to the chromatographic behavior of subtilisin in hydrophobic-interaction and reversed-phase chromatography.

Genetically engineered variants were used to examine how microenvironmental changes in the S1 substrate binding subsite of subtilisin contribute to chromatographic behavior of proteins on hydrophobic-interaction chromatography (HIC) and reversed-phase chromatography (RPC) columns. Gradient elution studies over a wide pH range showed that conditions could be found where a HIC support could separate proteins varying by one amino acid. Although all single-site variants could not be separated by HIC, this chromatographic mode was found to be complementary to cation-exchange chromatography for the separation of such variants. RPC was found to be of much less utility in the resolution of variant proteins. Retention and resolution of subtilisin variants was found to vary on RPC with the concentration and type of mobile phase pairing agent.

Amino Acids↗
PubMed1999

PfSUB-2: a second subtilisin-like protein in Plasmodium falciparum merozoites.

Erythrocyte invasion by the malaria merozoite requires the activity of merozoite proteases. We have previously identified a Plasmodium falciparum protein belonging to the superfamily of subtilisin-like serine proteases, which is expressed in a subset of secretory organelles in free merozoites. Here we describe the identification of a second P. falciparum subtilisin-like merozoite protein. Called PfSUB-2, it is encoded by a single copy gene and is expressed as a large putative type I integral membrane protein which undergoes extensive post-translational processing. The terminal processing product is expressed in an apical location in merozoites. PfSUB-2 may mediate one or more of the serine protease activities known to be associated with erythrocyte invasion.

Amino Acid Sequence↗
PubMed1998

Effect of pressure on the catalytic activity of subtilisin Carlsberg suspended in compressed gases.

We studied the effect of pressure up to 300 bar on the catalytic efficiency of subtilisin Carlsberg suspended in compressed propane, near-critical ethane, near-critical carbon dioxide and tert-amyl alcohol, at constant temperature and fixed enzyme hydration. Increasing pressure lowered the catalytic efficiency of the enzyme in all the solvents, resulting in positive activation volumes, delta V#. The delta V# values in compressed propane and in tert-amyl alcohol were similar and larger in magnitude than the value reported in the literature for the same reaction in an aqueous buffer, although within the range of typical delta V# values in aqueous media. In the near-critical fluids, the delta V# were much larger, e.g., an increase in pressure of only 200 bar causing a sixfold decrease in the catalytic efficiency of subtilisin in carbon dioxide. These data should reflect the proximity of ethane and carbon dioxide to the critical point, and the resulting condensation of solvent molecules about the solutes, yielding negative solute partial molar volumes.

Carbon Dioxide↗
PubMed2001

Peptide synthesis in organic media with subtilisin 72 immobilized on poly(vinyl alcohol)-cryogel carrier.

Serine proteinase subtilisin 72 was covalently attached to the beads of poly(vinyl alcohol)-cryogel, a macroporous hydrogel prepared by the freeze-thaw technique. The immobilized enzyme was examined as a catalyst in the synthesis of protected peptides Z-Ala-Ala-Xaa-Phe-pNA (Xaa = Leu, Glu, Lys) in acetonitrile/dimethylformamide mixtures. Immobilized subtilisin catalyzed with high yield the formation of peptide bonds between Phe-pNA and acyl donors including those with free carboxylic group and non-protected C-terminal basic and acidic amino acid residues.

Acetonitriles↗
PubMed1996

A hyperthermostable protease of the subtilisin family bound to the surface layer of the archaeon Staphylothermus marinus.

BACKGROUND: Staphylothermus marinus, an archaeon isolated from a geothermally heated marine environment, is a peptide-fermenting, sulphur-dependent organism with an optimum growth temperature of 92 degrees C. It forms grapes of cells, which adhere to each other and to sulphur granules via their surface layer. This glycoprotein layer forms a canopy which is held at a distance of about 70 nm from the cell membrane by membrane-anchored stalks, thereby enclosing a 'quasi-periplasmic space'. Two copies of a globular protease, which probably serves an exodigestive function related to the organism's energy metabolism, are attached near the middle of each stalk. RESULTS: We have purified and characterized this protease with regard to its enzymatic properties and thermostability, and have sequenced its gene using an approach based entirely on the polymerase chain reaction. The precursor form is 1345 amino acids long; between residues 64-741, it contains a domain with clear homology to subtilisins, which is interrupted by two large insertions. The enzyme has a broad substrate specificity and a pH optimum of 9.0. It is fully stable from pH 3.2 to 12.7 and is resistant to heat-inactivation to 95 degrees C in the free form and to 125 degrees C in the stalk-bound form. CONCLUSIONS: This protease is one of the most stable proteases known. Its high resistance towards denaturing agents makes it an interesting target for practical applications. Despite its large size, it is clearly a member of the subtilisin family and represents the only known enzyme that is a stoichiometric S-layer component.

Amino Acid Sequence↗
PubMed1995

The prosegment-subtilisin BPN' complex: crystal structure of a specific 'foldase'.

BACKGROUND: The folding of the bacterial protease subtilisin BPN' (SBT) is dependent on its 77-residue prosegment, which is then autocatalytically removed to give the mature enzyme. Mature subtilisin represents a class of proteins that lacks an efficient folding pathway. Refolding of mature SBT is extremely slow unless catalyzed by the independently expressed prosegment, leading to a bimolecular complex. RESULTS: We report the crystal structure at 2.0 A resolution of the prosegment-SBT complex and consider its implications for prosubtilisin BPN' maturation and folding catalysis. The prosegment forms a compact domain that binds SBT through an extensive interface involving the enzyme's two parallel surface helices (residues 104-116 and 133-144), supplying negatively charged caps to the N termini of these helices. The prosegment C terminus binds in the enzyme active site in a product-like manner, with Tyr77 in the P1 binding pocket. CONCLUSIONS: The structure of the complex supports a unimolecular mechanism for prosubtilisin cleavage, involving a 25 A rearrangement of the SBT N terminus in a late folding step. A mechanism of folding catalysis in which the two helices and their connecting beta strand form a prosegment-stabilized folding nucleus is proposed. While this putative nucleus is stabilized by prosegment binding, the N-terminal and C-terminal subdomains of SBT could fold by propagation.

Crystallography, X-Ray↗
PubMed2003

Interaction of GroEL and GroEL/GroES complexes with a nonnative subtilisin variant: a small-angle neutron scattering study.

Small-angle neutron scattering and contrast variation were used to study the solution structure of GroEL and GroEL/GroES chaperonins complexed with a nonnative variant of the polypeptide substrate, subtilisin (PJ9). The subtilisin was 86% deuterated (dPJ9) so that it contrasted sufficiently with the chaperonin, allowing the contrast variation technique to be used to separate the scattering from the two components bound in the complex. Both the native double-ring GroEL and a single-ring mutant were used with dPJ9 bound in a 1:1 stoichiometry per GroEL toroid. This allowed both the position and the shape of dPJ9 in the GroEL/dPJ9 complexes to be determined. A single-ring GroEL/GroES variant complexed with one dPJ9 molecule was used to study the structural changes of dPJ9 in GroEL/GroES/dPJ9 complexes formed with ADP and with ATP. It was found that both the shape and the position of the bound dPJ9 in the GroEL/GroES/dPJ9 complex with ADP were the same as those in the GroEL/dPJ9 complex. However, dPJ9 assumed a more symmetric shape when bound in the GroEL/GroES/dPJ9 complex with ATP. This important observation reflects the relative ability of ATP to promote refolding of protein substrates relative to that of ADP.

Adenosine Diphosphate↗