Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests.

The major storage proteins in cereals, prolamins, have an abundance of the amino acids glutamine and proline. Storage pests need specific digestive enzymes to efficiently hydrolyze these storage proteins. Therefore, post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored-product pest, Tenebrio molitor (yellow mealworm). Three distinct PGP activities were found in the anterior and posterior midgut using the highly-specific chromogenic peptide substrate N-benzyloxycarbonyl-L-Ala-L-Ala-L-Gln p-nitroanilide. PGP peptidases were characterized according to gel elution times, activity profiles in buffers of different pH, electrophoretic mobility under native conditions, and inhibitor sensitivity. The results indicate that PGP activity is due to cysteine and not serine chymotrypsin-like peptidases from the T. molitor larvae midgut. We propose that the evolutionary conservation of cysteine peptidases in the complement of digestive peptidases of tenebrionid stored-product beetles is due not only to the adaptation of insects to plants rich in serine peptidase inhibitors, but also to accommodate the need to efficiently cleave major dietary proteins rich in glutamine.

[Properties of post-proline cleaving enzymes from Tenebrio molitor].

Two post-proline cleaving enzymes PRE1 and PRE2 with molecular masses of 101 and 62 kDa, respectively, capable of hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the midgut of the flour beetle Tenebrio molitor and characterized. PRE1 is active only in acidic media, with a maximum at pH 5.6, whereas PRE2, both in acidic and alkaline media with a maximum at pH 7.9. Using inhibitory analysis, both PRE1 and PRE2 were shown to belong to serine peptidases. Some data indicate that a Cys residue is located close to the PRE2 active site. Z-Pro-prolinal, a specific inhibitor of prolyl oligopeptidases, inhibits completely PRE2 and partially PRE1. The substrate specificities of the isolated enzymes were studied. It was shown that Z-AlaAla-Pro-pNA was the best substrate for PRE1, and Z-AlaPro-pNA, for PRE2. The combination of the studied properties allowed characterization of PRE2 as a prolyl oligopeptidase.

[Chemoenzymatic synthesis of new fluorogenous substrates for cysteine proteases of the papain family].

A chemoenzymatic syntheses was developed for new highly specific fluorogenic substrates for cysteine proteases of the papain family, Abz-Phe-Ala-pNA (I) and Glp-Phe-Ala-Amc (II) (Abz, pNA, Glp, and Amc are i-aminobenzoyl, p-nitroanilide, pyroglutamyl, and 4-amino-7-methylcoumaride, respectively). Substrate (I) was obtained in an aqueous-organic medium using native chymotrypsin. Substrate (II) was synthesized in DMF-MeCN by the treatment with chymotrypsin and subtilisin Carlsberg immobilized on polyvinyl alcohol cryogel. Hydrolysis of substrate (I) with papain, ficin, and bromelain was accompanied by a 15-fold increase in fluorescence intensity, and that of substrate (II), by a change in the fluorescence spectrum. Unambiguity of enzymatic hydrolysis of the substrates after the Ala residue was shown. The specific activity of the substrate hydrolysis with papain, bromelain, and ficin and was determined. Papain showed the greatest activity for both substrates. The activity of all proteases under study was essentially higher for substrate (II), than for substrate (I). The lowest detectable papain concentrations were 2.4 x 10(-10) M for (I) and 1.2 x 10(-11) M for (II). A high selectivity of cysteine proteases for Glp-Phe-Ala-Amc was established.

[Biocatalytic properties of thermolysin immobilized on polyvinyl alcohol cryogel].

Preparations with different contents of thermolysin were obtained by the immobilization of the enzyme on granulated polyvinyl alcohol cryogel. Their activity and stability in an aqueous medium and in mixtures of polar organic solvents of different composition were investigated. The catalytic properties of the preparations in reactions of peptide bond formation were studied, and the optimal amount of the biocatalyst, the concentrations of initial reagents, and the ratios of organic solvents and water necessary for effective enzymatic peptide synthesis catalyzed by immobilized thermolysin were determined. A series of peptides of the general formula Z-Ala-Ala-Xaa-pNA, where Xaa = Leu, Ile, Phe, Val, or Ala, were synthesized, and the immobilized enzyme was shown to retain substrate specificity in an organic medium.

[Native and modified subtilisin Karlsberg as an effective catalyst of peptide bond formation in organic media].

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z- ALA-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P1 and P'1 (Glu, Arg, and Asp) containing unprotected side ionogenic groups.

[Peptide synthesis in organic media with the use of subtilisin 72 immobilized on a poly(vinyl alcohol) cryogel].

Subtilisin 72 serine protease (EC 3.4.21.14) immobilized on a poly(vinyl alcohol) cryogel was used as a catalyst in the syntheses of N-protected peptide p-nitroanilides of the general formulas Z(or Boc)-Xaa-Phe-pNA (Xaa = Leu or Ala), Z-Ala-Xaa-Yaa-pNA (Xaa = Leu or Ala; Yaa = Leu or Phe), and Z-Ala-Ala-Xaa-Yaa-pNA (Xaa = Leu, Arg, or Gly; Yaa = Phe, Leu, Gly, Asp, or Glu). The syntheses were carried out in DMF-acetonitrile mixtures. A number of protected di-, tri-, and tetrapeptides were prepared in yields up to 99%. The syntheses were found to retain stereoselectivity under the conditions studied. The activation of carboxyl group of the acylating component was shown to have a positive effect upon the coupling rate. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

Activity and stability of native and modified subtilisins in various media.

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.

[Native and modified subtilisin 72 as a catalyst of peptide synthesis in media with low water content]. / Nativnyi i modifitirovannyi subtilizin 72 kak ktalizator sinteza peptidov v sredakh s nizkim soderzhaniem vody.

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.

[The enzymatic segment condensation of peptides on a solid phase in organic medium]. / Fermentativnaia blochnaia kondensatsiia peptidov na tverdoi faze v organicheskoi srede.

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 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-A and Dnp-Ala-Ala-Leu-Ala-Ala-Glu(OMe)-Ala-Ala-Leu-Met-Ala-Gly-A (A is 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.

Fluorogenic substrates for assay of chymosin.

The use of fluorogenic substrates with intramolecular fluorescence quenching as substrates for chymosin was studied. It was shown that chymosin hydrolyzes the Phe-Phe peptide bond. The effect of pH on the hydrolysis of substrates by chymosin was investigated. The catalytic characteristics of the hydrolysis of the fluorogenic substrates were obtained at the pH optima. The influence of dimethylformamide on chymosin activity was studied.

[A study of aspartyl proteases using intramolecularly quenched fluorogenic peptide substrates]. / Peptidnye substraty s vnutrimolekuliarnym tusheniem flyorestsentsii dlia izucheniia aspartil'nykh proteinaz.

A series of fluorogenic tetra-, penta-, and hexapeptide substrates of the general structure Abz-X-Phe-Phe-Y-Ded (or -pNa in place of -Ded), where X = Ala, Ala-Ala, or Val-Ala and Y = -, Ala, or Ala-Ala, were proposed. Kinetic parameters of hydrolysis of these substrates by pepsin, cathepsin D, human gastricsin, pig pepsin, calf chymosin, and aspergillopepsin A were determined. The compounds synthesized proved to be effective substrates for aspartyl proteases of diverse origins.

[Peptide synthesis catalyzed by subtilisin-72 in organic solvents]. / Peptidnyi sintez, kataliziruemyi subtilizinom-72 v organicheskikh rastvoriteliakh.

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

[Synthesis of three- and tetrapeptides catalyzed by subtilisin suspensions in organic solvents]. / Sintez tri- i tetrapeptidov, kataliziruemyi subtilizinom v vide suspenzii v organicheskikh rastvoriteliakh.

The behavior of subtilisin 72 in some aprotic solvents (acetonitrile, dioxane, and tetrahydrofurane) was studied. The enzyme was shown to be partially soluble in tetrahydrofurane, but it is rendered profoundly inactive in this solution. In acetonitrile and dioxane, subtilisin formed dilute suspensions whose activities were measured after dilution with water. Under these conditions, subtilisin suspended in acetonitrile manifested an activity that was an order of magnitude higher than that of its dioxane suspension, and this activity continued for a long time. Z-Ala-Ala-Leu-pNA was synthesized from Z-Ala-Ala-OCH3 and Leu-pNA under the catalysis by dilute suspension of subtilisin in acetonitrile. p-Nitroanilides of tetrapeptides, Z-Ala-Ala-P1-P'1-pNA, where P1 and P'1 were either Leu or Phe, were similarly synthesized in acetonitrile under catalysis by dilute subtilisin suspension at [S]:[E] = 10(5):1. p-Nitroanilides of tripeptides, Z-Ala-Ala-Leu-pNA, Z-Ala-Ala-Phe-pNA, and Z-Ala-Ala-Phe-NH2, were also synthesized in the presence of a concentrated subtilisin suspension at [S]:[E] = 10(3):1. It was shown that the increase in enzyme concentration resulted in the double coupling of nucleophile, and Z-Ala-Ala-Leu-Leu-pNA, Z-Ala-Ala-Phe-Phe-pNA, and Z-Ala-Ala-Phe-Phe-NH2 were obtained with 13, 33, and 40% yields, respectively. Therefore, such reaction systems can be used for creating long hydrophobic peptides whose synthesis in water-organic mixtures is difficult due to the poor solubility of starting components in aqueous buffer solutions.

Thiolsubtilisin as an instrument for peptide synthesis. Preparation and properties.

A convenient procedure for thiolsubtilisin purification from an admixture of subtilisin involving affinity chromatography on bacitracin-Sepharose is presented. Thiolsubtilisin activity was measured by hydrolysis of p-nitrophenyl acetate, p-nitroanilide-peptide (Glp-Ala-Ala-Leu-pNA), and azocasein. The thiolenzyme catalyzes peptide synthesis. Under these conditions only activated peptide esters, e.g., p-chlorophenyl, N-hydroxysuccinimide, or p-nitrophenyl esters form peptide bonds during interaction with appropriate nucleophiles such as peptides and their derivatives and amino acid amides.

Fluorogenic peptide substrates for assay of aspartyl proteinases.

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 and N-(o-aminobenzoyl-Ala-Ala-Phe-Phe-Ala-Ala)-N'-2,4-dinitrophenyl ethylenediamine, contain a fluorescent o-aminobenzoyl moiety as well as p-nitroaniline or N-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 of o-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.

Proteinase-catalyzed peptide synthesis in concentrated solutions of urea and other denaturing agents.

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 were 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 hydrochloride 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.

[Synthesis of peptides in organic solvents catalyzed by pepsin]. / Kataliziruemyi pepsinom sintez peptidov v organicheskikh rastvoriteliakh.

The porcine pepsin immobilized on inorganic supports catalyzes the peptide bond formation in organic solvents. Dependence of the peptide bond formation between Z-Ala-Ala-Phe-OH and H-Leu-Ala-Ala-OCH3 upon the porous material, organic solvent, reaction time, enzyme concentration, ionic strength and pH was studied. Syntheses of peptides of the general formula Z-Ala-Ala-Xaa-Yaa-Ala-Ala-OCH3, where Xaa = Phe, Tyr, Trp; Yaa = Leu, Phe, Tyr, Trp, were carried out.

[Co-precipitation of pepsin with products from the enzymatic synthesis of peptides as a factor limiting the effectiveness of the enzyme]. / Soosazhdenie pepsina s produktami fermentativnogo sinteza peptidov kak faktor, ogranichivaiushchii éffektivnost' fermenta.

Porcine pepsin behaviour during the synthesis of peptide p-nitroanilides and esters has been studied. In many cases, especially when long-chain peptides, such as Z-Ala-Ala-Phe-Leu-Ala-Ala-OMe, were synthesized, pepsin disappeared from the solution, being entrapped by the product precipitate rather than inactivated. Sorption of the enzyme on the product might be partially responsible for this effect. The active enzyme could be eluted from the precipitate by NaCl and isopropanol. Non-proteolytic proteins (lysozyme, bovine albumin, carbonic anhydrase) could also co-precipitate with pepsin.

[Pepsin in the enzymatic synthesis of esters and n-nitroanilide peptides]. / Pepsin v fermentativnom sintexze éfirov i n-nitroanilidov peptidov.

Pepsin was shown to catalyze synthesis of esters or p-nitroanilides tri-, tetra-, penta- and hexapeptides of general formula Z-X-Y-B, where X = Ala-Phe, Phe-Met, Ala-Ala-Glu, Ala-Ala-Phe, Ala-Ala-Leu, Ala-Ala-Trp, Ala-Ala-Met. Y = Ala, Leu, Val, Phe, Arg, Ala-Ala, Gly-Gly, Leu-Ala-Ala, Phe-Ala-Ala. B = OMe, pNA. The reactions were carried out in dimethylformamide-water solutions at pH 4.6 by equimolar ratio of amino- and carboxyl components (with the exception of Arg-pNA taken in 2-fold excess). The amount of pepsin in the reaction approached 1:1700 enzyme: substrate molar ratio although it might be improved--up to 1:3.10(5) for relatively long peptides.

[Serine proteinase from the archaebacterium Halobacterium mediterranei--an analog of eubacterium subtilisin]. / Serinovaia proteinaza arkhebakterii Halobacterium mediterranei--analog subtilizina éubakterii.

A homogeneous serine proteinase was isolated from cultural filtrates of the extreme halophilic bacteria Halobacterium mediterranei 1538 using affinity chromatography on bacitracin-Sepharose, ultrafiltration and gel filtration on Sephadex G-75, with a 48% yield and 260-fold purification. The enzyme was completely inactivated by specific inhibitors of serine proteinases, PMSF and DFP, as well as by Hg2+ and PCMB. The enzyme activity was strongly dependent of NaCl concentration, the enzyme being inactivated below 0.75 M NaCl. Inactivation of the enzyme was also seen in the presence of 2-7% organic solvents. The pH optimum for Glp-Ala-Ala-Leu-pNA hydrolysis is 8.0-8.5; Km is 0.14 mM, kcat is 36.9 s-1. The stability optimum lies at pH 5.5-8.0, temperature optimum is at 55 degrees C. The enzyme molecular weight is 41,000 Da; pI is 7.5. The substrate specificity of the enzyme is comparable to that of secretory subtilisins; the extent of protein substrate hydrolysis is similar to that of proteinase K. The N-terminal sequence of Halobacterium mediterranei serine proteinase, Asp-Thr-Ala-Asn-Asp-Pro-Lys-Tyr-Gly-Ser-Gln-Tyr-Ala-Pro-Gln-Lys-Val-Asn- Ala- Asp-, reveals a 50% homology with the aminoterminal sequence of Thermoactinomyces vulgaris serine proteinase. Hence, the serine proteinase secreted by halophilic bacteria may be considered as a structural and functional analog of eubacterial enzymes.