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Swine pepsin at pH 5 efficiently catalyzes a condensation between Z-Ala-Ala-Phe-OH and p-nitroanilides of Leu, Phe, Val, Ala and Arg that leads to formation of corresponding benzyloxycarbonyl-tetrapeptide p-nitroanilides with yields of 70-90%. These reactions are complicated by co-precipitation of pepsin and the reaction products that necessitates the use of a relatively high concentration of pepsin.

Pepsin successfully catalyzed the synthesis of several hydrophobic octa- and decapeptides in dimethylformamide-water solutions containing concentrated urea at pH 4.65. The factors that influence peptide synthesis in the presence of urea aere studied using condensation of the tripeptides Z-Ala-Ala-Phe-OH and H-Leu-Ala-Ala-OCH3 as a model. The dependence of Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 yield on pepsin concentration and pH, as well as the behavior of pepsin during peptide synthesis were studied. It was shown that pepsin catalyzed the synthesis of Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 in guanidine hydro-chloride and sodium dodecyl sulfate solutions. Other proteinases, subtilisin and thermolysin, were applied for the synthesis of p-nitroanilides of tri- and tetrapeptides in urea solutions. Proteinase-catalyzed peptide synthesis in the presence of denaturing agents might help to overcome the limitations caused by poor solubility of the starting peptide derivatives, although this effect is sometimes counterbalanced by the product solubility. ©Munksgaard 1996.

The segment condensation of peptides on a solid phase (Aminosilochrom) in organic medium catalyzed by a subtilisin complex with sodium dodecylsulfate was studied. The dependence of the efficiency of the enzymatic coupling of tripeptides with the basic structure X-Ala-Ala-Y-OMe [where X = Z, Boc, or Dnp and Y = Leu or Glu(OMe)] on the spacer (Phe-Met-Gly-Gly) content on the support and on the structure of the acylating component was investigated. The tripeptide segments were successively coupled to Aminosilochrom containing the Met-Ala-Gly spacer, and the peptidylaminosilochroms Dnp-Ala-Ala-Leu-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-Aand Dnp-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A(Ais the Aminosilochrom residue) were obtained in satisfactory yields. It was shown by these examples that the second and third segments are attached in yields higher than that for the first segment and the coupling efficiency does not depend on the amino acid composition of the acylating component.

The subtilisin-sodium dodecyl sulfate complex was shown to catalyze the coupling of peptide segments on a solid phase in organic medium. By a two-stage enzymic condensation of peptide fragments on aminosilochrom (A) containing Met-Ala-Gly as a spacer, Dnp(or Boc)-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Met-Ala-Gly-A and Z-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A were obtained. It was shown that the condensation products can be split off from the support using Met residue cleavage by BrCN.

Bacillus thuringiensis "true" toxins consist of three domains: the N-terminal, alpha-helical domain followed by two beta-structural domains. Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains. There are at least two patterns of the limited proteolysis of "true" toxins. The first pattern, observed for CryIA and CryIVD delta-endotoxins, results in the proteolysis of the loops connecting beta-strands of the second domain. The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth alpha-helices of the first domain. The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase. Bioassay of CryIG and CryIVB delta-endotoxin fragments indicates that only two alpha-helices, the sixth and the seventh within the first domain, followed by the two beta-structural domains are sufficient for the insecticidal activity.

The structural gene of the carboxypeptidase T (cpt) was successfully expressed in cell wall-less L-form cells of Proteus mirabilis. The DNA sequence encoding the PhoA leader peptide was fused with a truncated cpt gene encoding the mature enzyme. The modified gene in a pUC-based kanamycin resistance vector under the control of the lac promoter was transformed into L-form cells of P. mirabilis. They were able to produce the recombinant CpT both as a secretory and as a cell-bound insoluble form. The co-secretory processing of the PhoA leader peptide was quite efficient. The yield of the secreted CpT was not less than 20 mg l-1 and should be improvable.

An enzymatic synthesis of tripeptide Z-Ala-AIa-Leu-pNA, tetrapeptides Z-Ala-Ala-P1-PI'- Xaa, where Pl = Leu, Trp, Met, Ala, lie, Phe; Pl' = Phe, Ala, Leu; Xaa = pNA, NH2, pentapeptides Z-Ala-Ala- Leu-Ala-Ala-pNA and Z-Ala-Ala-Leu-Ala-Phe-pNA is described. The reactions were performed in organic solvents using SDS-subtilisin complex as a catalyst of the peptide bond synthesis. © 1997 Elsevier Science Ltd. The use of proteases as catalysts of peptide synthesis is a promising direction of modem peptide chemistry. In a kinetically controlled process of peptide bond formation from peptide esters and aminocomponents one of the major obstacles is a secondary hydrolysis of the product. Peptide synthesis in organic solvents with low water content helps to solve this problem.

A new cryI-related sequence designated cryIM was cloned using an immunoscreening technique from ssp. wuhanensis of Bacillus thuringiensis (BT), previously reported to produce multiple Cry proteins [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026–1034]. Analysis of the cryIM nucleotide sequence revealed an ORF, BTII-type promoter-like sequence, peculiar for such genes, a translation initiation element and a putative transcription terminator. Nevertheless, its product was not previously found in the crystals of the host strain [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026–1034] which shows its weak or absent natural expression. The amino acid sequence of 1151 residues encoded by the continuous reading frame of cryIM is not identical but is essentially similar to the other δ-endotoxins of the CryI class. An IS231-like sequence was found 400 bp downstream of the cryIM stop codon and a fragment of the cryIAb gene was located 3 kb upstream of its initiator codon in the same orientation. Artificial expression of the cloned gene in E. coli under the control of the lacZ promoter allowed us to obtain its hypothetical protein product. © 1997 Federation of European Biochemical Societies.

A homogeneous glutamyl endopeptidase splitting peptide bonds of glutamic, rarely of aspartic acid residues in peptides and proteins, was isolated from Bacillus intermedius 3-19 culture filtrate using chromatography on CM cellulose and Mono S. The enzyme molecular mass is equal to 29 kDa, pI 8.4. The protease is inhibited by diisopropylfluorophosphate. The enzyme, like other glutamyl endopeptidases, reveals two pH optima at pH 7.5 and 9.0 for casein and one at pH 8.0 for Z-Glu-pNA hydrolysis. A 6 mM K m is found for hydrolysis of the latter substrate. The enzyme activity optimum lies at 55°C, and it is stable at pH 6.5–11.0. Its N-terminal sequence shows 56% coinciding residues when compared with that of Bacillus licheniformis glutamyl endopeptidase. © 1997 Federation of European Biochemical Societies.

An enzymatic synthesis of peptidyl amino alcohols is described. The reactions were performed in organic solvents using as a catalyst subtilisin distributed on the surface of macroporous silica. Subsequent mild oxidation of peptidyl amino alcohols results in peptide aldehydes — the potent specific inhibitors of serine proteinases.

A synthetic peptide derivative,—alanyl-alanyl-leucylmorpholide, (Ala-Ala-Leu-N(CH2CH2)2O), is suggested as a specific ligand for affinity chromatography of subtilisin-like serine proteases that prefer hydrophobic amino acid residue in P1 position of their substrates. Z-Ala-Ala-Leu-N(CH2CH2), a weak and reversible inhibitor of serine proteases, was synthesized by the carbodiimide method. Affinity sorbents were prepared by coupling the synthesized pepitide derivates to CH or AH Sepharose. Serine proteases from different sources were purified up to 17 fold on these sorbents with yields varying from 25% to 100%. Three enzymes (serine protease X, kallikrein and leucine aminopeptidase) were isolated from urine of children with glomerulonephritis with yields of 57, 22 and 55%. Proteolytic enzymes from the dandelion root, Kamchatka crab and culture filtrates of different microorganisms were also purified on the affinity sorbents.

Five chitinases were isolated from culture filtrates of Bacillus licheniformis B-6839 R and S variants by combination of hydrophobic, ion-exchange, and gel permeation chromatography. The enzymes had molecular masses of 66, 62, 53, 49, and 42 kDa. The chitinases revealed two activity optima against colloidal chitin at pH 4.5–5.5 and 9.0–9.5 and they were rather stable at pH 4.0–9.5. The temperature optimum of activity was 90 °C for the 62-kDa chitinase and 70 °C for the other enzymes. The 66-, 53-, and 42-kDa chitinases showed pronounced similarities in their N-terminal sequences and apparently belonged to the same group, which might be related to Bacillus circulans chitinase A1. The 49- and 62-kDa enzymes did not reveal structural similarities with other chitinases produced by the studied B. licheniformis strain. No relationship was found with the 89- and 76-kDa chitinases isolated earlier from B. licheniformis X-7u.Key words: Bacillus licheniformis, chitinase, multiplicity.

Via a combination of chemical and enzymatic synthesis, new hexapeptide substrates convenient for use in activity assessment of several aspartyl proteinases—porcine pepsin, human pepsin, gastricsin, and cathepsin D—were prepared. These peptide derivatives,o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala-p-nitroanilide andN-(o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala)-N′-2,4-dinitrophenyl ethylenediamine, contain a fluorescento-aminobenzoyl moiety as well asp-nitroaniline orN-2,4-dinitrophenyl ethylenediamine—the groups that cause fluorescence quenching. Aspartyl proteinases hydrolyze the Phe-Phe peptide bond in the substrates, which diminishes quenching due to separation of the fluorescent and quenching moieties and leads to an increase in the fluorescence intensity ofo-aminobenzoyl residue. Abz-Ala-Ala-Phe-Phe-Ala-Ala-Ded, being fairly well hydrolyzed by HIV proteinase, might be used for assay of this enzyme.

Buffalo chymosin was isolated from abomasum mucosa extract of buffalo calves by affinity chromatography on gramicidin S-agarose followed by ion exchange chromatography on gamma-aminopropylsilochrom. Its molecular weight, 36 +/- 1 kDa, is similar to that of bovine calf chymosin. The N-terminal sequence Gly-Glu-Val-Ala-Ser-Val-Pro- coincides with that of bovine enzyme, whereas some differences were found in the amino acid composition of these enzymes. Buffalo and bovine enzyme possess similar but not identical structures. General proteolytic and milk-clotting activities of buffalo chymosin are also similar to those of bovine proteinase. pH-Optimum of its activity against hemoglobin lies at pH 4.0, somewhat higher than that for bovine chymosin, which indicates subtle differences in the functional properties of two enzymes.

An enzymatic synthesis of Ac-Tyr-Ile-Gly-Ser-Arg-OH, its amide and p -nitroanilide starting with Boc-Ile-Gly-Ser-OCH 3 is described. The reactions were performed in organic solvents using subtilisin and α-chymotrypsin sorbed on macroporous silica surface as the catalysts.

Denaturation of Bacillus thuringiensis CryIIIA delta-endotoxin--an insecticidal protein, active against Coleoptera larvae--in concentrated guanidine hydrochloride solutions was pursued by fluorescence and circular dichroism spectroscopy and limited proteolysis. It was found that the protein consists of two fragments that differ by their stability to denaturation by guanidine hydrochloride at pH 3. The less stable fragment corresponds to the N-terminal alpha-helical domain limited by Leu-279; the more stable one starts with Ile-280, contains about 330 amino acid residues, and corresponds to the molecule C-terminal moiety that consist of its two beta-structural domains forming a superdomain.

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 α-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 α-chymotrypsin as a catalyst for peptide synthesis is more limited due to restricted specificity of this enzyme.

A method was developed to assess the number of δ-endotoxins contained in Bacillus thuringiensis entomocidal crystals. It utilized proteolytic conversion of 130-kDa protoxin into 60- to 65-kDa "true" toxin via limited proteolysis with trypsin and separation of stable N-terminal domains by fast-performance liquid chromatography. Immunodiffusion experiments and N-terminal sequence determination (applied to the major component isolated by SDS-PAGE) completed the analysis of the crystal protein composition. The application of this approach to crystals produced by cells of B. thuringiensis subsp. galleriae and wuhanensis allowed us to identify at least seven and eight different δ-endotoxins, respectively. Among those δ-endotoxins assigned to previously described families, CryIA, CryID, Cry IF, and CryIG were found, as well as crystal proteins, which possess N-terminal amino acid sequences very different from those of all known δ-endotoxins. Possible functional consequences of δ-endotoxin multiplicity are discussed.Key words: Bacillus thuringiensis, δ-endotoxins, multiplicity of crystal proteins, separation of true toxins.

A cry-related sequence, designated cryX (EMBL X75019), was localized upstream of cryIG, the δ-endotoxin gene cloned from spp. galleriae of Bacillus thuringiensis and sequenced earlier [(1991) FEBS Lett. 293, 25-28]. Analysis of the cryX complete nucleotide sequence enabled us to explain its virtual crypticity and to reveal the chimeric structure of the genes, cryX and cryIG. The amino acid sequence of 1,151 residues encoded by the continuous reading frame of cryX is similar to the other δ-endotoxins but differs essentially from them.

An enzymatic synthesis of peptidyl amino alcohols is described. The reactions were performed in organic solvents using as a catalyst subtilisin distributed on the surface of macroporous silica. Subsequent mild oxidation of peptidyl amino alcohols results in peptide aldehydes — the potent specific inhibitors of serine proteinases.

The primary structure of carboxypeptidase T--a Zn-dependent extracellular enzyme of Thermoactinomyces vulgaris--was determined from the cloned cpT gene nucleotide sequence and compared to Zn-carboxypeptidases from various organisms. The compilation and analysis of multiple alignment accompanied by consideration of available tertiary structure data have shown that in the overall spatial structure and active site arrangement CpT is similar to other enzymes constituting the Zn-carboxypeptidase family. Nine of 16 amino acid residues found to be strictly invariant are presumably located close to the active site. The preservation of His69, Glu72, Asn144, Arg145, His196, Tyr248, and Glu270 identified previously as essential catalytic site participants implicates basically the same catalytic mechanism in the Zn-carboxypeptidase family. It is proposed that Pro205 and Asp256 should play an important role in proper S1'-pocket spatial arrangement. The comparative analysis of amino acid variations in S1'-pocket enabled us to reveal structural determinants of the Zn-carboxypeptidase primary specificity. The relatively reduced size of the pocket and negative charge of Asp253 are supposed to contribute correspondingly to A- and B-type substrate preferences of carboxypeptidase T endowed with dual primary specificity.

The crystal structure of carboxypeptidase T from Thermoactinomyces vulgaris has been determined at 0.235-nm resolution by X-ray diffraction. Carboxypeptidase T is a remote homologue of mammalian Zn-carboxypeptidases. In spite of the low degree of amino acid sequence identity, the three-dimensional structure of carboxypeptidase T is very similar to that of pancreatic carboxypeptidases A and B. The core of the protein molecule is formed by an eight-stranded mixed beta sheet. The active site is located at the C-edge of the central (parallel) part of the beta sheet. The structural organization of the active centre appears to be essentially the same in the three carboxypeptidases. Amino acid residues directly involved in catalysis and binding of the C-terminal carboxyl of a substrate are strictly conserved. This suggests that the catalytic mechanism proposed for the pancreatic enzymes is applicable to carboxypeptidase T and to the whole family of Zn-carboxypeptidases. Comparison of the amino acid replacements at the primary specificity pocket of carboxypeptidases A, B and T provides an explanation of the unusual 'A+B' type of specificity of carboxypeptidase T. Four calcium-binding sites localized in the crystal structure of carboxypeptidase T could account for the high thermostability of the protein.

A gene coding for an extracellular Zn-carboxypeptidase of Thermoactinomyces vulgaris has been cloned and sequenced (EMBL X56901). This enzyme named carboxypeptidase T reveals simultaneously both types of substrate specificity characteristic of mammalian carboxypeptidases A and B. The carboxypeptidase T gene is primarily expressed in E. coli as a non-active preproenzyme with an additional 98 amino acid residues at the N-terminus. Primary structure alignment or mature carboxypeptidase T and mammalian metallocarboxypeptidases demonstrated 25–30% overall identity but a full preservation of presumed catalytically important residues. These observations imply a basic uniformity of the general catalytic mechanism for enzymes of that class produced by evolutionarily remote organisms.

N-Terminal domain (65 kD) of delta-endotoxin produced by Bacillus thuringiensis ssp. alesti, as shown by limited proteolysis, consists of two subdomains of molecular mass 30 and 33 kD that correspond, respectively, to conservative and variable regions of the delta-endotoxin primary structure. Furthermore, proteolysis of these subdomains leads to their conversion into at least two fragments of molecular mass 10 kD stable to proteinase action. Such a pattern of molecular organization appears to be common for several structurally related delta-endotoxins that belong to the kurstaki group. Entomicidal protein produced by ssp. israelensis (70 kD), which differs strongly from alesti and other kurstaki group delta-endotoxins, retains a similar type of molecular organization and consists of two subdomains with molecular mass of approximately 35 kD. Apparently, the characteristic pattern of the delta-endotoxins' molecular structure reflects separation of functions (e.g., host recognition and toxicity per se) between domains and subdomains of these proteins.

Publisher Summary This chapter examines the structural chemistry and the biological aspects of carboxypeptidase T. Activity of carboxypeptidase T is measured against chromogenic substrates: Dnp-Ala-Ala +Arg or Dnp-Ala-Ala-Leu + Arg or their analogs. This method is also suitable for assay of carboxypeptidase B or other carboxypeptidases capable of cleaving the C-terminal arginine from the substrates. Splitting of the C-terminal arginine changes the solubility of the Dnp moiety; the chromophore can be extracted into ethyl acetate from acidified aqueous solution, whereas the noncleaved substrate remains in the aqueous phase. An even simpler protocol, especially suitable for carboxypeptidase T assay in multiple samples, is based on an ion-exchange separation of the colored hydrolysis product from the excess of the substrate. The molecular mass of the single-chain enzyme is 36 928 Da based on the amino acid sequence and its pi is 5.3. The primary structure of carboxypeptidase T determined by DNA sequencing of the cloned structural gene agreed with the N-terminal sequence of 31 amino acid residues established by automated Edman degradation of the mature enzyme. The mature enzyme sequence consists of 326 amino acid residues, preceded by 98 residues that comprise the prepro region.

Two ways for semi-enzymatic preparation of the peptide aldehydes are proposed: (1) enzymatic acylation of amino alcohols with acyl peptide esters and subsequent chemical oxidation of the resulting peptide alcohols with DMSO/acetic anhydride mixture or (2) enzymatic acylation of the preliminarily obtained by a chemical route amino aldehyde semicarbazones. Subtilisin 72, serine proteinase with a broad specificity, distributed over macroporous silica, was used as a catalyst in both cases. Due to the practical absence of water in the reaction mixtures the yields of the products in both enzymatic reactions were nearly quantitative. The second way seems to be more attractive because all chemical stages were carried out with amino acid derivatives, far less valuable compounds than peptide ones. A series of peptide aldehydes of general formula Z-Ala-Ala-Xaa-al (where Xaa-al = leucinal, phenylalaninal, alaninal, valinal) was obtained. The inhibition parameters for these compounds, in the hydrolysis reactions of corresponding chromogenic substrates for subtilisin and alpha-chymotrypsin, were determined.

The glutamyl endopeptidase gene of Bacillus intermedius was cloned from a genomic library expressed in Bacillus subtilis and sequenced (EMBL accession number Y15136). The encoded preproenzyme contains 303 amino acid residues; the mature 23-kDa enzyme consists of 215 residues. The mature enzyme reveals 38% of identical residues when aligned with the glutamyl endopeptidase from Bacillus licheniformis, whereas only five invariant residues were found among all known glutamyl endopeptidases. The amino acid residues that form the catalytic triad (H47, D98, and S171) as well as H186 participating in the binding of the substrate carboxyl group were identified. It seems that the structural elements responsible for the function of glutamyl endopeptidases from various sources are highly variable.

A serine proteinase was isolated from fruits of Maclura pomifera (Raf.) Schneid. by affinity chromatography on bacitracin-containing sorbents and gel-filtration. The enzyme, named macluralisin, is a glycoprotein with a molecular mass of 65 kDa; its protein moiety corresponds to a molecular mass of 50 kDa. The substrate specificity of macluralisin towards synthetic peptides and insulin B-chain is similar to that of cucumisin, a subtilisin-like proteinase from melon fruit. The enzyme is completely inhibited by diisopropylfluorophosphate. Its amino-acid composition resembles that of a serine proteinase isolated from the Cucurbitaceae. The N-terminal sequence has 33% of its residues identical to those of the sequence of fungal subtilisin-like proteinase K. Hence, Maclura pomifera serine proteinase belongs to the subtilisin family, which seems to be broadly distributed in the plant kingdom.

The progress of enzymatic peptide synthesis catalyzed by alpha-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH2 (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous silica material. All amino acid residues were of the L-configuration. The solvent consisted of acetonitrile, dimethylformamide (DMF), and 4% (v/v) of water. The DMF/acetonitrile ratio was varied between 0 and 1/1. At high concentration of the acyl-group donor and approximately equimolar ratio of the nucleophile and the acyl-group donor, quantitative formation of MalAlaAlaPheXaaNH2 or ZAlaAlaPheXaaNH2 occurred. As a result, a method for the synthesis of polypeptide amides was developed. At low concentration of the acyl-group donor and excess of the nucleophile, the condensation by-products with two and three nucleophile molecules were found in the reaction mixtures. The data obtained provided evidence that organic solvents affected the S'1-specificity of alpha-chymotrypsin and the S1-specificity of subtilisin 72, while the S1-specificity of alpha-chymotrypsin and the S'1-specificity of subtilisin 72 were not affected. When the DMF content was increased, the rate of the alpha-chymotrypsin-catalyzed reactions decreased. In contrast to this, an increase in DMF content accelerated the subtilisin 72-catalyzed reactions. Hydrolysis of the acyl-group donor did not occur in the alpha-chymotrypsin-catalyzed reactions. Significant (up to 50%) formation of MalAlaAlaPheOH was observed at the early stage of the subtilisin 72-catalyzed reactions. Later MalAlaAlaPheOH underwent synthesis.

alpha-Chymotrypsin-catalyzed acyl transfer was studied using three acyl-group donors (Mal-L-Ala-L-Ala-L-PheOMe, Bz-L-TyrOEt and Ac-L-TrpOEt; Mal, maleyl; Bz, benzoyl; OMe, methyl ester; OEt, ethyl ester) and a series of amino-acid amides. Most of the reactions studied can be described by the simplest kinetic model without the nucleophile binding to the acyl-enzyme. The alpha-chymotrypsin-catalyzed transfer of the Mal-L-Ala-L-Ala-L-Phe group to the amides of L-Phe and L-Tyr showed a linear dependence of the partition constant, p, on the nucleophile concentration which can be interpreted by the hydrolysis of the acyl-enzyme-nucleophile complex. The alpha-chymotrypsin-catalyzed transfer of the Bz-L-Tyr and Ac-L-Trp groups to several amino-acid amides showed unusual behavior which can be interpreted by the kinetic model involving formation of a complex of the acyl-enzyme with two nucleophile molecules. These observations can explain the conflicting conclusions concerning the kinetics of alpha-chymotrypsin-catalyzed acyl transfer evident in previous studies.

In α-chymotrypsin-catalyzed acyl-transfer reactions in water the specificity of the enzyme (the nucleophile reactivity of amino acid amides) is correlated with the substrate hydrophobicity and increases as the hydrophobicity of the side chain of the amino acid amides is increased. In a low water system (4% H2O) bulky amino acid amides are less efficient nucleophiles. The specificity of α-chymotrypsin towards the amino acid amides in acyl transfer reactions in this case does not depend on the hydrophobicity of the amino acid side chains but correlates with their size. Therefore, different factors can be responsible for the specificity of enzymes in water and in a mainly organic medium.

A homogeneous serine proteinase secreted by the extreme halophilic bacterium Halobacterium mediterranei 1538 was isolated by affinity chromatography on bacitracin-Sepharose with a yield of 48% (260-fold purification). The enzyme reveals an optimum for pyroglutamyl-Ala-Ala-Leu p-nitroanilide hydrolysis at pH 8.0-8.5 (Km 0.14 mM; k(cat). 36.9 s-1). Its activity increases linearly with NaCl concentration over the range 2-5 M. The substrate specificity of the enzyme is comparable with that of secretory subtilisins, the extent of protein degradation approaching that attained with proteinase K. The enzyme has a molecular mass of 41 kDa and a pI of 7.5. The N-terminal sequence of H. mediterranei serine proteinase reveals a 50% identity with that of Thermoactinomyces vulgaris serine proteinases, indicating that the enzyme belongs to the subtilisin family. Hence the serine proteinase secreted by the halophilic bacterium should be considered as a functional analogue, and a structural homologue, of eubacterial serine proteinases (subtilisins).

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.

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.

A gene cryIg coding for entomocidal protein delta-endotoxin of Bacillus thuringiensis ssp. galleriae str. 11-67 named CryIG has been cloned and sequenced (EMBL accession number X58120). The deduced amino acid sequence that contains 1156 amino acid residues shows only 28% of identical residues, when compared with other delta-endotoxins of the CryI family. The extent of identity is substantially higher for some regions of the sequence ('conserved blocks'), that presumably bear important structural or functional properties. This implies that CryIG delta-endotoxin follows the same type of polypeptide chain folding as other CryI proteins, whereas peculiarities of primary structure help to explain its unique specificity.

Prochymosin can be converted into chymosin by an action of external proteinases. Thus, thermolysin at pH 5.05 converts calf prochymosin into active Phe-chymosin, which is one amino acid longer than chymosin from the N-terminus with a yield of 73%. Even better results were achieved with prochymosin activation by Legionella pneumophila metalloproteinase. Apparently the stretch of prochymosin polypeptide chain adjacent to the normally observed activation point becomes available for an attack by an external proteinase at pH 5.0–6.0. These data indicate that the intermolecular activation pathway might be of physiological importance.

Summary Intracellular serine protease was isolated in a pure state from sporulating Bacillus subtilis A-50. The enzyme has the molecular weight about 30.000 daltons and pI 4.3, is completely inhibited with phenylmethylsulfonyl fluoride and EDTA, and possesses a rather low activity against protein substrates, but high specific activity with subtilisin chromogenic substrates. Amino acid composition shows higher level of Glx, Lys and Phe residues and lower content of Val residues in comparison with known subtilisins. N-terminal sequence of the enzyme, being two residues shorter from N-terminus than subtilisin BPN', is NH 2 - Ser -Leu- Pro -Glu- Gly -Ile-X-Val- Ile-Lys-Ala-Pro -Glu- Leu -Gln-Ala- Gln-Gly -Phe-Lys-(residues identical with BPN' are underlined). This clearly indicates the presence of two homologous structural genes, for intra- and extracellular serine proteases, in B.subtilis genome.

1. It has been shown that polypeptide antibiotics — bacitracin and gramicidin S — inhibit carboxylic proteinases — porcine and equine pepsins. Bacitracin inhibits pepsin with KI=2.3 mM, and gramicidin S with KI∼2 mM.

The crystalline proteins fromBac. thuringiensis of the third and fourth serotypes with a molecular weight of 130,000 have been isolated and carboxymethylated, and their amino acid compositions has been determined. The CM-proteins have been found to be labile in acid media (80% CF3COOH and 65% HCOOH). Conditions have been found for the cleavage of the CM-protein of the third serotype with cyanogen bromide. A BrCN fragment with a molecular weight of 16,000 has been isolated, the intensity of which in 5% polyacrylamide gel on scanning amounts to about half the intensity of the sum of all the fragments. The aminoacid composition of this fragment has been determined.

A sorbent obtained by treatment of cyanogen bromide-activated Sepharose 4B with mono-N-DNP-hexamethylenediamine has been shown to be effective in the affinity chromatography of pepsin, pepsinogen and acid proteinase from Aspergillus awamori. It is considered that 2,4-dinitrophenyl residues of the sorbent interact specifically with the hydrophobic zone of the enzyme, which may belong to the substrate binding site. The chromatography of chymotrypsin on the same sorbent supports this assumption.

A new polymeric sorbent for proteinases has been synthesized by the radical copolymerization of N-vinylpyrrolidone, bis-Nδ-acryloylgramicidin C, and N,N′-methylenebisacrylamide. Biospecific chromatography on the new sorbent has enabled an industrial preparation of procine pepsin to be purified by a factor of 2.5. With the aid of the new sorbent, a carboxylic proteinase has been isolated from the industrial preparation Tsellolignorin with a 15-fold purification factor.

1. 1. From the cultural filtrate of Acremonium chrysogenum—producer of antibiotic cephalosporin C—by isopropyi alcohol precipitation and affinity chromatography on bacitracin-Sepharose two serine proteinases (I and II) were isolated. 2. 2. Both enzymes of mol. wt 28,000 were inhibited by phenylmethylsulfonylfluoride, diisopropylfluorophosphate, ovomucoid and antipain. Proteinase I was inhibited by p -hydroxymercuribenzoate, whereas Hg 2+ ions suppressed the activity of both proteinases. 3. 3. The pH-optimum of the enzymes activity against Z-Ala-Ala-Leu- p NA lies at pH 10-10.5. 4. 4. pi of proteinase I exceeds 10.0, that of proteinase II is equal to 4.0. 5. 5. The enzymes differ by pH-dependence of their stability as well as by their action on protein and synthetic peptide substrates. 6. 6. The N-terminal sequences revealed clear cut homology between these proteinases which are presumed to belong to the specific group of serine proteinases remotely related to bacterial subtilisins.

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA)--a convenient chromogenic substrate for assay of thiol proteinases papain, ficin, and bromelain--was prepared by enzymatic synthesis with chymotrypsin as a catalyst. The thiol proteinases hydrolyze PFLNA with the liberation of p-nitroaniline, estimated spectrophotometrically by its absorbance at 410 nm. The phenylalanine residue in the P2 position of PFLNA meets the specificity demands of thiol proteinases. The following values of Km were found for PFLNA hydrolysis: by papain, 0.34 mM; by ficin, 0.43 mM; by bromelain, 0.30 mM. This substrate was successfully applied to monitor thiol proteinase affinity chromatography on bacitracin-Sepharose, which resulted in a 2- to 4-fold purification from commercial preparations.

Via attachment of p-(omega-aminoethyl)phenylboronic acid to CH-Sepharose in the presence of water-soluble carbodiimide, a new sorbent for the biospecific chromatography of serine proteinases was obtained. The sorbent was shown to be suitable for the purification of subtilisn, alpha-chymotrypsin and trupsin. It is assumed that the serine hydroxyl group at the active site of the enzyme forms, with the boronic acid moiety of the ligand, a structure that imitates transition enzyme--substrage complex. The presence of glycerol selectively improves the binding of serine proteinases, presumably because of stabilization of the tetrahedral state of the boron atom. Direct isolation of subtilisin from a Bacillus subtilis cultural filtrate on phenylboronic acid-containing sorbent gives a virtually homogenous enzyme (42-fold purification) in a nearly-quantitative yield.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.