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PubMed1985

Thermodynamics of the binding of Streptomyces subtilisin inhibitor to alpha-chymotrypsin.

The binding of Streptomyces subtilisin inhibitor (SSI) to alpha-chymotrypsin (CT) (EC 3.4.21.1) was studied by isothermal and differential scanning calorimetry at pH 7.0. Thermodynamic quantities for the binding of SSI to the enzyme were derived as functions of temperature from binding constants (S. Matsumori, B. Tonomura, and K. Hiromi, private communication) and isothermal calorimetric experiments at 5-30 degrees C. At 25 degrees C, the values are delta G degrees b = -29.9 kJ mol-1, delta Hb = +18.7 (+/- 1.3) kJ mol-1, delta S degrees b = +0.16 kJ K-1 mol-1, and delta C p,b = -1.08 (+/- 0.11) kJ mol-1. The binding of SSI to CT is weak compared with its binding to subtilisin [Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 1195-1202; Takahashi, K., & Fukada, H. (1985) Biochemistry 24, 297-300]. This difference is due primarily to a less favorable enthalpy change in the formation of the complex with CT. The hydrophobic effect is presumably the major source of the entropy and heat capacity changes which accompany the binding process. The unfolding temperature of the complex is about 7 degrees C higher than that of the free enzyme. The enthalpy and the heat capacity changes for the unfolding of CT were found to be 814 kJ mol-1 and 17.3 kJ K-1 mol-1 at 49 degrees C. The same quantities for the unfolding of the SSI-CT complex are 1183 kJ mol-1 and 39.2 kJ K-1 mol-1 at 57 degrees C.

Bacterial Proteins↗
PubMed1989

High-level expression in Streptomyces lividans 66 of a gene encoding Streptomyces subtilisin inhibitor from Streptomyces albogriseolus S-3253.

A secretory expression system for Streptomyces subtilisin inhibitor (SSI) was established in a heterologous host, Streptomyces lividans 66, by introducing the 1.8-kbp BglII/SalI fragment containing SSI gene into the Streptomyces multicopy vector, pIJ 702. The expression of SSI did not depend on the orientation of the 1.8-kbp BglII/SalI fragment or on the promoter for tyrosinase gene (mel) in pIJ 702, which suggested that this fragment also carries the SSI promoter. The expressed SSI in S.lividans 66 was secreted into the culture medium in a large amount, as observed with the original strain, S. albogriseolus S-3253. Amino acid sequence analysis showed that the SSI secreted from S. lividans 66 contained three additional amino acid residues in the NH2-terminal region. The inhibitory activity toward subtilisin BPN' and the antigenic activity of the SSI secreted from S. lividans 66 were found to be identical with those of authentic SSI.

Amino Acid Sequence↗
PubMed1982

Dynamic states of the three methionyl residues of Streptomyces subtilisin inhibitor. 1H NMR studies.

High resolution 1H NMR spectroscopy at 360 and 400 MHZ was used to study the dynamic states of the three methionyl residues (Met 70, 73, and 103) of Streptomyces subtilisin inhibitor (SSI) in a neutral aqueous (2H2O) solution. Chemical modification and nuclear Overhauser effect studies show that one of the methyl groups exists in the interior of the protein and is surrounded by phenylalanyl residues. With reference to the crystal structure, this methyl group is assignable to that of Met 103 in the hydrophobic core. The other two methionyl residues (Met 70 and 73) are equally susceptible to chemical modifications, but their methyl signals are affected in a different manner by the alpha-chymotrypsin-cleavage of the peptide bond between Met 73 and Val 74 at the reactive site. The observed linewidths of the methyl groups are consistent with the assumption that the whole side-chain of both Met 70 and 73 are already undergoing rapid internal motions at 20 degrees C, whereas for Met 103 only the rotation of the methyl group is allowed, even above approximately 40 degrees C. The high-field shift of the methyl proton resonance of Met 103 by about 0.3 ppm is attributable to the ring current effect from the surrounding phenylalanyl residues. This shift is invariant in the temperature range of 40-90 degrees C, showing that the native environment of the methyl group of Met 103 persists up to 90 degrees C at neutral pH. Destruction of the native methionyl environments occurs only above 90 degrees C, and the rate of conversion between the native and the denatured conformations is slower than 10 s-1. When SSI is mixed with subtilisin BPN' in a one-to-one molar ratio, at least one of the methyl signals of Met 70 and 73 is completely broadened, showing that the side-chain of this methionine is directly involved in the interaction with the enzyme.

Bacterial Proteins↗
PubMed1989

Isolation and Partial Characterization of a Subtilisin Inhibitor from the Mung Bean (Vigna radiata).

The subtilisin inhibitor (MBSI-A) from the mung bean (Vigna radiata (L.) Wilczek) seed has been purified to homogeneity. MBSI-A consists of a single polypeptide chain of 119 residues, with a high content of glutamic acid/glutamine, aspartic acid/asparagine, valine, threonine, and proline (19, 12, 10, 9, and 8 residue percent, respectively). MBSI-A is a potent inhibitor of subtilisin Carlsberg, but is inactive toward bovine trypsin and alpha-chymotrypsin and the plant cysteinyl proteinase papain. The MBSI is located exclusively in the cytosol of the seed cotyledon cell, unlike the mung bean trypsin inhibitor (MBTI), which is located primarily in the protein bodies. Both MBSI and MBTI accumulate in the seed during the most active period of reserve protein accumulation, 12 to 18 days after flowering. During germination MBSI, like MBTI, is broken down beginning 2 to 3 days after seed imbibition. The disappearance of MBSI-A is accompanied by the transient appearance of a new inhibitor species, MBSI-D. The amino acid composition of MBSI-D suggests that it may be produced by the loss of approximately 20 amino acid residues from MBSI-A.

Journal Article↗
PubMed1994

Packing selection of Bacillus lentus subtilisin and a site-specific variant.

The crystallization of a variant of Bacillus lentus subtilisin and the native enzyme was achieved using identical conditions. The variant B. lentus was found to grow in two crystal forms, form 1 and form 2, whereas the native B. lentus subtilisin enzyme crystallized in only one, form 1. Form 2 crystals, once obtained, were found to grow much more rapidly than form 1 crystals. The lattice contacts and structural changes giving both crystal forms have been examined. The results show that crystal form 2 has a more complex network of interactions. There is also a small surface conformational change in the form 2 structure relative to the native and variant form 1 crystals and at least two solvent molecules bound to the enzyme in crystal form 1 are displaced in crystal form 2. In addition, a site specific substitution in the variant at position 27 induces a 'short' lattice contact which does not exist in the native B. lentus or the form 2 variant B. lentus. These results suggest that in some circumstances engineered variants could be designed to crystallize more rapidly than the native enzyme.

Journal Article↗
PubMed1993

Streptomyces subtilisin inhibitor-like proteins are distributed widely in streptomycetes.

Streptomyces subtilisin inhibitor-like proteins were found to be distributed widely in streptomycetes by using the combination of the convenient, newly developed plate assay system and an established liquid culture assay. Almost all the strains formerly categorized as Streptoverticillium species produced proteins that exhibited inhibitory activity against both subtilisin BPN' and trypsin. N-terminal regions of three purified proteins showed high structural similarity to those of other previously reported SIL inhibitors.

Amino Acid Sequence↗
PubMed1971

Inhibition of the plaquing efficiency of T4r+ bacteriophage by subtilisin.

The mechanism by which the replicative cycle of T4r(+) phage is inhibited by certain nonhost bacterial systems was investigated. Some Bacillaceae, especially Bacillus subtilis, decreased the plaquing efficiency of this virus more than 95% within 24 hr of exposure. Sarcina lutea and Micrococcus sp. both failed to cause any significant change in the infectivity of T4r(+) phage. Preliminary investigations into the nature of the inhibitory substance(s) suggested that an extracellularly elicited protein was at least partially responsible for this effect. Further analysis has implicated subtilisin, an exoprotease from B. subtilis, as the cause of some, if not all, of the observed decrease in plaquing efficiency. Gel-filtration chromatography of control and treated (14)C-labeled T4r(+) phage showed a wide dispersal of phage-specific material of these particles after 24 hr of exposure to pure subtilisin or to expended medium exoprotease from B. subtilis. It was concluded that B. subtilis exoprotease is capable of chemically altering the structure of the phage capsid, thus causing a decrease in its plaquing efficiency.

Amylases↗
PubMed2000

Application of a metal switch to aqualysin I, a subtilisin-type bacterial serine protease, to the S3 site residues, ser102 and gly131.

We applied 'metal switch' experiments to the S3 site residues, Ser102 and Gly131, of aqualysin I, a subtilisin-type serine protease. We showed that two histidines introduced at these positions did take part in histidine-metal-histidine bridge formation, and metal ions inhibited the protease activities. These results indicate that two histidines are near each other, and both side chains are metal-accessible. This is the first report on application of the metal-switch technique to a subtilisin-related enzyme.

Amino Acid Substitution↗
PubMed1998

Purification and characterization of a 315 kDa keratinolytic subtilisin-like serine protease from Microsporum canis and evidence of its secretion in naturally infected cats.

A keratinolytic protease, secreted as the major component by a feline clinical isolate of Microsporum canis cultivated in a minimal medium containing cat keratin, was purified by affinity chromatography on bacitracin agarose and gel filtration. The apparent molecular mass of the enzyme was 31.5 kDa and the pI was 11.8. The enzyme was not glycosylated and its first 15 N-terminal amino acids showed numerous similarities with other fungal subtilisins. The optimum pH was around 9 while inactivation of the enzyme was reversible at pH 4, but not at pH 11. The enzyme was stable at 37 degrees C with an apparent optimum temperature around 55 degrees C. PMSF, soybean trypsin inhibitor (SBTI) and chymostatin strongly inhibited the proteinase. The highest affinity (Km of 0.37 mM) and physiological efficiency (k(cat)/Km) were obtained for the synthetic substrate N-Suc-Ala-Ala-Pro-Phe-p-nitroanilide. These results indicate that the keratinase belongs to the subtilisin-like serine protease family. Purified rabbit immunoglobulins G prepared against the keratinase and used in an immunohistochemical test allowed the detection of the keratinase produced by the fungus invading hair structures in naturally infected cats. The in vitro keratinolytic activity of the enzyme and its production in vivo suggest that it may contribute to pathogenicity.

Amino Acid Sequence↗
PubMed1998

Dynamic structure of subtilisin-eglin c complex studied by normal mode analysis.

Normal mode analysis of subtilisin-eglin c complex was performed to investigate the dynamics at the interface between the enzyme and the inhibitor. The internal motions of the complex calculated from the normal modes were divided into three parts: the internal motions changing the shape of each molecule, the external rigid-body motions changing their mutual dispositions, and the coupling between the internal and external motions. From the results of the analysis, the following characteristic features were found in the dynamics at the interface regions: 1) negative correlation between the internal and external motions within each molecule, and 2) positive correlation between the external motions of the two molecules. The former decreases the apparent amplitudes of motions at the interface. The latter minimizes the interference between individual motions of the two molecules. These dynamic characteristics allow the enzyme and the inhibitor to move as freely as possible. This finding suggests that the experimental evidence of the large entropy gain on binding should be attributed not only to strong hydrophobic interactions, but also to the dynamic structure of the complex, which is found to minimize an unavoidable loss of the conformational entropy on binding.

Models, Molecular↗
PubMed1998

Solubilization of subtilisin in CO2 using fluoroether-functional amphiphiles.

Carbon dioxide is a naturally abundant, environmentally benign solvent whose use, like water, in a process is not regulated by either EPA or FDA. Unfortunately, polar compounds such as amino acids and proteins are essentially insoluble in carbon dioxide. Further, alkyl-functional surfactants, which have been shown to allow extraction of proteins into conventional organic solvents, exhibit very poor or negligible solubility in CO2 at pressures below 50 MPa. Consequently, highly CO2-soluble fluoroether-functional surfactants have been generated and used to solubilize subtilisin Carlsberg from aqueous buffer and cell culture medium into CO2, with recovery accomplished by depressurization. Both the amount of protein solubilized in the emulsion and the extent of activity retention by the protein following recovery are functions of the initial protein concentration in the buffer. This, plus the observation that the presence of protein affects the stability of the emulsion, suggests that some of the protein is sacrificed to act as a stabilizer in these systems. In addition to solubilization via an inverse emulsion, it has also been shown that one can strip protein-surfactant aggregates from a middle phase emulsion using pure CO2, suggesting an ion-pairing type mechanism.

Biotechnology↗
PubMed1997

Murine subtilisin-like proteinase SPC6 is expressed during embryonic implantation, somitogenesis, and skeletal formation.

During mammalian embryogenesis, proteinases are important for both matrix remodeling and the activation of latent growth factors. As subtilisin-like prohormone convertases (SPCs) have recently been found to activate members of the matrix metalloproteinase and transforming growth factor-beta (TGF-beta) families, we sought to investigate the role of this gene family in murine implantation and embryogenesis. Using active site polymerase chain reaction (PCR) cloning, four members of the SPC family were identified at embryonic day 6.5: SPC1, SPC2, SPC3, and SPC6. In situ hybridization analysis of sectioned E6.5 embryos in utero demonstrated strong SPC6 expression in differentiated decidua, overlapping and extending beyond the region previously described for the metalloproteinase inhibitor TIMP-2. Lower levels of SPC6 expression were observed in trophoblasts and in the ectoplacental cone, suggesting multiple roles for this enzyme in implantation. Northern analysis showed that SPC6 mRNA in embryos is represented by two distinct sizes of message--the isoform SPC6-1 (3.0 kb) is most abundant at all stages, but significant levels of SPC6-b (6.0 kb) occur in E12.5 embryos. Whole mount in situ hybridization to E8.5 embryos demonstrated strong SPC6 expression in the most posterior somite. This somitic staining moved caudally with the developing embryo and by E10.5 became localized to the posterior of the tail, indicating that SPC6 is involved in somitogenesis. SPC6 was expressed at low levels throughout the embryo, except in the developing nervous system, and strong expression was observed in the first branchial arch and in skeletal regions of the developing vertebrae, limbs, and craniofacium, suggesting additional roles for SPC6 in skeletogenesis.

Animals↗
PubMed2002

Chemically modified "polar patch" mutants of subtilisin in peptide synthesis with remarkably broad substrate acceptance: designing combinatorial biocatalysts.

A significant enhancement of the applicability of the serine protease subtilisin Bacillus lentus (SBL) in peptide synthesis was achieved by using the strategy of combined site-directed mutagenesis and chemical modification to create chemically modified mutant (CMM) enzymes. The introduction of polar and/or homochiral auxiliary substituents, such as X=oxazolidinones, alkylammonium groups, and carbohydrates at position 166 at the base of the primary specificity S(1) pocket created SBL CMMs S166C-S-X with strikingly broad structural substrate specificities. These CMMs are capable of catalyzing the coupling reactions of not only L-amino acid esters but also D-amino acid esters as acyl donors with glycinamide to give the corresponding dipeptides in good yields. These powerful enzymes are also applicable to the coupling of L-amino acid acyl donors with alpha-branched acyl acceptor, L-alaninamide. Typical increases in isolated yields of dipeptides of 60-80 % over SBL-WT (e.g. 0 % yield of Z-D-Glu-GlyNH(2) using SBL-WT-->74 % using S166C-S-(CH(2))(2) NMe(3) (+)) demonstrate the remarkable synthetic utility of this "polar patch" strategy. Such wide-ranging systems displaying broadened and therefore similarly high, balanced yields of products (e.g. 91 % Z-L-Ala-GlyNH(2) and 86 % yield of Z-D-Ala-GlyNH(2) using S166C-S-(3R,4S)-indenooxazolidinone) may now allow the use of biocatalysts in parallel library synthesis.

Bacillus↗
PubMed2001

Variations in the enantioselectivity of salt-activated subtilisin induced by lyophilization.

Including excess salt during lyophilization has been shown to increase the activity of freeze-dried subtilisin Carlsberg (SC) in anhydrous media by over 20,000-fold [Ru et al. (1999) Biotechnol Bioeng 63:233-241]. In the present study, salt-activated SC (KCl-SC) showed a 30% enhancement in enantioselectivity compared to the salt-free enzyme in a variety of organic solvents. Activity toward both enantiomers of N-acetyl-phenylalanine methyl ester (APME) increased in tandem by 2-3 orders of magnitude in all solvents, indicating that the mechanism of salt activation is inherent to the enzyme and does not strongly favor one enantiomer over the other. However, activity and enantioselectivity of salt-activated SC could be manipulated through changes in the lyophilization conditions. Variations in lyophilization time, initial KCl concentration, and initial lyophilization volume altered enantioselectivity over 2-fold. The changes in enantioselectivity reflected the activity for the L enantiomer, while the activity toward the D enantiomer was mostly unaffected. The results indicate that the lyophilization time and final water content of the KCl-SC are important determinants of enzyme activity for the L enantiomer, suggesting that the favored reaction is more sensitive to the structural integrity of the salt-activated enzyme.

Freeze Drying↗
PubMed1984

Three-dimensional structure of fungal proteinase K reveals similarity to bacterial subtilisin.

The three-dimensional structure of the fungal serine protease proteinase K has been determined at 3.3 A resolution by single crystal X-ray diffraction analysis. The enzyme crystallizes in the tetragonal space group P4(3)2(1)2 with cell constants a = b = 68.3 A, c = 108.5 A. The asymmetric unit consists of one monomer of 27 000 daltons mol. wt., approximately 50% higher than the so far assumed value of 18 500 daltons. The main chain fold of proteinase K shows a high degree of tertiary homology with the corresponding bacterial subtilisin BPN'. Proteinase K is the second enzyme in this family of serine proteases to be studied by X-ray diffraction, thus confirming the existence of two unrelated families of serine proteases in pro-and eukaryotes.

Bacteria↗
PubMed1992

Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease.

Many viruses have membrane glycoproteins that are activated at cleavage sites containing multiple arginine and lysine residues by cellular proteases so far not identified. The proteases responsible for cleavage of the hemagglutinin of fowl plague virus, a prototype of these glycoproteins, has now been isolated from Madin-Darby bovine kidney cells. The enzyme has a mol. wt of 85,000, a pH optimum ranging from 6.5 to 7.5, is calcium dependent and recognizes the consensus sequence R-X-K/R-R at the cleavage site of the hemagglutinin. Using a specific antiserum it has been identified as furin, a subtilisin-like eukaryotic protease. The fowl plague virus hemagglutinin was also cleaved after coexpression with human furin from cDNA by vaccinia virus vectors. Peptidyl chloroalkylketones containing the R-X-K/R-R motif specifically bind to the catalytic site of furin and are therefore potent inhibitors of hemagglutinin cleavage and fusion activity.

Affinity Labels↗
PubMed1990

Crystal structures of subtilisin BPN' variants containing disulfide bonds and cavities: concerted structural rearrangements induced by mutagenesis.

The X-ray structures of four genetically engineered disulfide variants of subtilisin have been analyzed to determine the energetic and structural constraints involved in inserting disulfide bonds into proteins. Each of the engineered disulfides exhibited atypical sets of dihedral angles compared with known structures of natural disulfide bridges in proteins and affected its local structural environment to a different extent. The disulfides located in buried regions, Cys26-Cys232 and Cys29-Cys119, induced larger changes than did Cys24-Cys87 and Cys22-Cys87, which are located on the surface of the molecule. An analysis of the concerted changes in secondary structure units such as alpha-helices and beta-sheets indicated systematic long-range effects. The observed changes in the mutants were largely distributed asymmetrically around the inserted disulfides, reflecting different degrees of inherent flexibility of neighboring secondary structure types. The disulfide substitution in each variant molecule created some invaginations or cavities, causing a reorganization of the surrounding water structure. These changes are described, as well as the changes in side chain positions of groups that border the cavities.

Base Sequence↗
PubMed2000

Highly regulated expression of subtilisin-like proprotein convertase PACE4 (SPC4) during dentinogenesis.

Expressions of mRNAs for four subtilisin-like proprotein convertases (SPCs: furin, PACE4, PC6, and PC8) and bone morphogenetic protein 4 (BMP4) in the rat molar tooth during development were analyzed by Northern blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization to explore the possible involvement of SPCs in the processing of proBMPs. We found a temporospacial expression of PACE4, but not one of the other SPCs, in this tissue; i.e., RT-PCR analysis revealed that the level of PACE4 mRNA, but not that of the other SPC mRNAs became high around the second postnatal day. This increase was in good accordance with the increase in BMP4 mRNA, indicating an apparent association of these molecules with the differentiation and establishment of functional ameloblasts and odontoblasts. During dentinogenesis, PACE4 mRNA was localized in the ameloblasts and odontoblasts. These observations suggest that PACE4 plays a crucial role in dentinogenesis, especially via the activation of BMPs.

Animals↗