Peptide diazomethyl ketones are inhibitors of subtilisin-type serine proteases

Peptide diazomethyl ketones, well known as specific cysteine protease inhibitors are also potent inhibitors of the microbial serine proteases thermitase (EC 3.4.21.14) and subtilisin Carlsberg (EC 3.4.21.14). The affinity of the enzymes towards the synthetic inhibitors Z-Ala(n)-PheCHN2 (n = 0, 1, 2) depends on the chain length and is in the same range as for the corresponding chloromethyl ketones. Both kinds of inhibitors react irreversibly in a 1:1 ratio with the enzymes and covalently bind to the active site histidine of both subtilisin Carlsberg and thermitase despite the fact that thermitase contains an active-site cysteinyl residue. The mechanism of the inhibition reaction is discussed.     

Preprosubtilisin Carlsberg processing and secretion is blocked after deletion of amino acids 97-101 in the mature part of the enzyme

Summary During an investigation into the substrate specificity and processing of subtilisin Carlsberg fromBacillus licheniformis, two major independent findings were made: (i) as has been shown previously, a stretch of five amino acids (residues 97–101 of the mature enzyme) that loops out into the binding cleft is involved in substrate binding by subtilisin Carlsberg. In order to see whether this loop element also determines substrate specificity, the coding region for these five amino acids was deleted from the cloned gene for subtilisin Carlsberg by site-directed mutagenesis. Unexpectedly the resulting mutant preproenzyme (P42c, M_r=42 kDa) was not processed to the mature form (M_r=30 kDa) and was not released into the medium by a proteasedeficientB. subtilis host strain; rather, it accumulated in the cell membrane. This result demonstrates that the integrity of this loop element, which is very distant from the processing cleavage sites in the preproenzyme, is required for secretion of subtilisin Carlsberg. (ii) In culture supernatants fromB. subtilis harbouring the cloned wild-type subtilisin Carlsberg gene the transient appearance (at 0–3 h after onset of stationary phase) of a processing intermediate (P38c, M_r=38 kDa) of this protease could be demonstrated. P38c very probably represents a genuine proform of subtilisin Carlsberg.     

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.     

A serine alkaline protease from the fungusConidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease fromConidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, theConidiobolus protease is structurally distinct from subtilisin Carlsberg. TheConidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. TheConidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.     

Effect of organic solvents on enantioselectivity of protease catalysis

The protease-catalyzed transesterifications between N-trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoroethyl ester and 1-propanol were studied in a variety of anhydrous organic solvents at 30 degrees C. The protease preparations lyophilized from phosphate buffer solutions (pH 8.0) were used as catalysts. The organic solvent affected both rate of reaction and enantioselectivity differently. Proteases such as Aspergillus oryzae protease, subtilisin Carlsberg, and subtilisin BPN' always preferred the L-enantiomer in both hydrophilic and hydrophobic solvents, indicating no inversion of the L-specificity in hydrophobic solvents such as toluene. However, enantioselectivity was rather poor, with E (enantiomeric ratio) values not exceeding even one order of magnitude except for acetonitrile. There was a weak inverse correlation between E values of subtilisin Carlsberg and solvent hydrophobicity (logP). Acetonitrile was a preferable solvent in terms of both rate of reaction and enantioselectivity (E= 15 to 25) for processing L-amino acid derivatives in organic media. Organic solvents generally have potential advantages of processing D-amino acid derivatives. (c) 1997 John Wiley & Sons, Inc.     

Crystal structure of an alkaline protease from Bacillus alcalophilus at 2.4 A resolution

The crystal structure of an alkaline protease from Bacillus alcalophilus has been determined by X-ray diffraction at 2.4 A resolution. The enzyme crystallizes in space group P2(1)2(1)2(1) with lattice constants a = 53.7, b = 61.6, c = 75.9 A. The structure was solved by molecular replacement using the structure of subtilisin Carlsberg as search model. Refinement using molecular dynamics and restrained least squares methods results in a crystallographic R-factor of 0.185. The tertiary structure is very similar to that of subtilisin Carlsberg. The greatest structural differences occur in loops at the surface of the protein.     

A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).     

Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus.

The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 A resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has been refined to an R-factor of 14.0% and contains 1882 protein atoms, two calcium ions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore, almost all of the secondary structure elements found in subtilisin Carlsberg are also present in the PB92 protease. The major differences between the two structures are located around the deletion regions (residues 37 and 158-161 in subtilisin Carlsberg) and in two loops which are known to be the most variable parts of subtilisin structures. Flexibility of one of these loops (residues 126-130 in the PB92 protease) is believed to account for the induced-fit mechanism of substrate binding.     

Peptide synthesis by proteases in organic solvents: medium effect on substrate specificity.

The substrate specificities of alpha-chymotrypsin and subtilisins for peptide synthesis in hydrophilic organic solvents were investigated. Chymotrypsin exhibited high specificity to aromatic amino acids as acyl donors, while subtilisin Carlsberg and subtilisin BPN' were specific to aromatic and neutral aliphatic amino acids, in accordance with the S1 specificities of the enzymes for peptide hydrolysis in aqueous solutions. On the contrary, chymotrypsin exhibited higher specificities to hydrophilic amino acid amides as acyl acceptors (nucleophiles) for peptide synthesis with N-acetyl-L-tyrosine ethyl ester, in contrast to the S1' specificity for peptide hydrolysis and peptide synthesis in aqueous solutions. Furthermore, nucleophile specificity changed with the change in water-organic solvent composition; the increase in water content led to increase in relative reactivity of leucinamide to that of alaninamide. It was also found that protection of the carboxyl group of alanine by amidation is much preferable to protection by esterification in terms of reactivity as nucleophiles.     

Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feathers

Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. Studies presented herein demonstrate that B. licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes from B. licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression of subE-encoded subtilisin from B. subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B. subtilis strain. While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.     

地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。     

Purification and characterization of subtilisin DJ-4 secreted by Bacillus sp. strain DJ-4 screened from Doen-Jang

Bacillus sp. strain DJ-4, which produces extracellular proteases, was screened from Doen-Jang, a traditional Korean fermented food. A fibrinolytic enzyme (subtilisin DJ-4) was purified using commercial chromatographic techniques. The relative molecular mass of the isolated protein was 29 kDa by SDS-PAGE and fibrin zymography assay. The enzyme was characterized as a serine protease by an inhibitor assay on the fibrin zymography gel and by an amidolytic assay using a chromogenic substrate. The enzyme was inhibited by PMSF, but not by EDTA or leupeptin. The first 14 amino acids of the N-terminal sequence were identical to that of subtilisin BPN', but the activity of subtilisin DJ-4 was 2.2 and 4.3 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively.     

Enzymatic reactions in aqueous-organic media. VIII. Medium effect and nucleophile specificity in subtilisin-catalyzed peptide synthesis in organic solvents

Summary Subtilisin Carlsberg and subtilisin BPN' (nagarse) catalyze peptide bond formation from aromatic amino acid esters and glycinamide in hydrophilic organic solvents. The activities of subtilisin and product compositions are different in several organic solvents; reactions in acetonitrile, tetrahydrofuran, and propylene carbonate gave the peptide in excellent yields, while in N,N-dimethylformamide and methanol the enzyme activity was largely retarded. The yield of the peptide is also dependent on water content in the reaction solutions. Optimum water contents are in the range from 3 to 7 %. The reaction is strongly specific for glycinamide as an amine component, and amides of alanine, valine, and leucine gave the corresponding peptides in poor yields.     

Subtilisin 72: a serine protease from Bac. subtilis strain 72 - an enzyme similar to subtilisin Carlsberg]

Subtilisin 72, a serine proteinase secreted by Bac. subtilis strain 72 was purified by covalent chromatography on Sepharose sorbent containing p-(omega-aminomethyl)phenylboronic acid as a ligand. The homogeneity of subtilisin 72 was confirmed by isoelectrofocusing in a thin layer of polyacrylamide gel (pl 8.6). The amino acid composition of this enzyme is different from that of other subtilisins, e. g. subtilisin Carlsberg. The N = terminal amino acid sequence of subtilisin 72 traced up to the 35th residue turned to be the same as that of subtilisin Carlsberg with the exception of the 21st (Tyr) and the 30th (Ile) residues. This very pronounced extent of homology shows that subtilisin 72 is very similar although not identical to subtilisin Carlsberg.     

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.     

Crystal and molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg

The crystal structure of the molecular complex of eglin, a serine proteinase inhibitor from leeches, with subtilisin Carlsberg has been determined at 2.0 A resolution by the molecular replacement method. The complex has been refined by restrained-parameter least-squares. The present crystallographic R factor (Formula: see text) is 0.183. Eglin is a member of the potato inhibitor 1 family, a group of serine proteinase inhibitors lacking disulfide bonds. Eglin shows strong structural homology to CI-2, a related inhibitor from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure from barley seeds. The structure of subtilisin Carlsberg in this complex is very similar to the known structure of subtilisin novo, despite changes of 84 out of 274 amino acids.     

The autocatalytic processing of the subtilisin Carlsberg pro-region is independent of the primary structure of the cleavage site

Subtilisins are extracellular seryl-proteases produced by bacilli (Markland and Emil, 1971). In addition to signal sequences, these proteases have N-terminal extensions (pro-regions) which have also been identified in several other proteases (Silen et al., 1988; Vasantha et al., 1984; Polhner et al., 1987; Henderson et al., 1987; Yanagida et al., 1986; Takagi et al., 1985). The pro-region holds the pro-protease associated with the membrane and release of the protease takes place as a result of pro-region removal by autocatalytic processing (Egnell and Flock, 1991). In this report we describe the construction of four deletion-mutations in the gene encoding subtilisin Carlsberg at the junction between the pro-region and mature subtilisin Carlsberg. We found that the introduction of different deletions abolished the ability of subtilisin to undergo autocatalytic cleavage of the pro-region in cis, whereas cleavage by exogenous subtilisin could still occur in trans. Point mutations were also introduced in positions -5 to +4 around the pro-region and native subtilisin cleavage site. Processing of pro-subtilisin with the point mutations showed that the autocatalytic cleavage and recognition of this junction of the subtilisin Carlsberg pro-region is independent of the amino acid sequence around the cleavage site.     

Enzymatic Oligomerization of the Tetrapeptide Ester Allylglycine-Phenylalanine-Phenylalanine-Allylglycine Ethyl Ester

The native hydrolytic action of subtilisin Carlsberg was reversed to oligomerize the tetrapeptide ester L-Ag-L-Phe-L-Phe-L-Ag-OEt, containing the unnatural amino acid allyglycine (Ag, where R = -CH₂-CH = CH₂), in several miscible aqueous/organic solvent systems. Mass spectrometry indicated that the octapeptide ester (Ag-Phe-Phe-Ag)² OEt was formed in all cases and that the dodecapeptide ester (Ag-Phe-Phe-Ag)₃OEt was formed in one case. Additional mass spectrometry peaks indicated that a number of other peptides larger than the octapeptide ester (Ag-Phe-Phe-A)²OEt may also have been formed. Infrared analysis revealed that the oligopeptide products exhibit P-sheet peptide folding.     

Bacillus licheniformis protease-catalyzed peptide synthesis via the kinetically controlled approach using the carbamoylmethyl ester as an acyl donor in anhydrous acetonitrile

The couplings of N-protected amino acid esters with amino acid amides proved to be carried out in anhydrous acetonitrile in the presence of Bacillus licheniformis protease (subtilisin Carlsberg) immobilized on Celite. The maximal peptide yields were obtained with the immobilized enzyme prepared through lyophilization from a pH 10.7 buffer solution. A series of dipeptide syntheses and several segment condensations were achieved generally in high yields by the combined use of the immobilized enzyme prepared from this pH and the carbamoylmethyl ester as the acyl donor.     

Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis.

The structural gene for a subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was determined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start site (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprised of 275 residues. The productivity of subtilisin in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence of the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69% identity with subtilisin Carlsberg, 89% with subtilisin BPN' and 70% with subtilisin DY. Some properties of the subtilisin J that had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. It retains about 50% of its activity even after treatment at 60 degrees C for 30 min in the presence of 2 mM calcium chloride.