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V M Stepanov

Publications and source records attributed to V M Stepanov.

At least 55 records · Page 3Linked to original sources
PubMed1994

[Primary structure of the intracellular serine proteinase from Bacillus amyloliquefaciens. III. Amino acid sequence of peptides obtained by hydrolysis with a Glu,Asp-specific proteinase. Reconstruction of the entire amino acid sequence of the proteinase].

Glu,Asp-specified protease hydrolysate of intracellular serine proteinase (ISP) was separated by ion-exchange chromatography on a sulphocationite resin followed by HPLC to yield 30 individual peptides. Their sequences, spanning to 243 amino acid residues, were determined by the manual Edman procedure. Four overlapping fragments were reconstructed by comparing their sequences with those of tryptic and chymotryptic peptides. To arrange these fragments in the proteinase polypeptide chain and to reconstruct the enzyme's total sequence, additional peptides were isolated from the tryptic hydrolysate and analysed. Primary structure of ISP, corresponding to 297 amino acid residues, was reconstructed. Its comparison with related serine proteinases revealed the following levels of homology: with Bacillus subtilis intracellular serine proteinase, 88%; with secretory subtilisin BPN' produced by B. amyloliquefaciens, 46%.

Amino Acid Sequence↗
PubMed1994

[Proteinases with various substrate specificities in structural studies of yeast cell walls].

Different proteins are revealed in cell wall of yeast cells Candida utilis by means of specific proteolysis with subtilisins TV and 72, trypsin and purified collagenase of Clostridium histolyticum. Some of them were characterized by resistance to trypsin and sensitivity to subtilisin TV. In young cells this group is represented essentially by a protein of 33 kD, which appears to be one of the structural proteins, binding fibrillae of carbohydrate. Other proteins proved to be sensitive to both trypsin and subtilisin. Among these proteins a protein with mol. mass 80 kD was revealed; its sensitivity to extremely specific hydrolysis by bacterial collagenase suggests it to contain amino acid sequences characteristic for collagens of higher eukaryotes.

Amino Acid Sequence↗
PubMed1994

[Synthesis of peptides in organic solvents catalyzed by pepsin].

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.

Acetates↗
PubMed1993

Primary structure of cryX**, the novel delta-endotoxin-related gene from Bacillus thuringiensis spp. galleriae.

A cry-related sequence, designated cryX (EMBL X75019), was localized upstream of cryIG, the delta-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 delta-endotoxins but differs essentially from them.

Amino Acid Sequence↗
PubMed1993

The second nucleophile molecule binds to the acyl-enzyme-nucleophile complex in alpha-chymotrypsin catalysis. Kinetic evidence for the interaction.

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.

Acylation↗
PubMed1993

Synthesis of tetrapeptide p-nitroanilides catalyzed by pepsin.

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.

Amino Acid Sequence↗
PubMed1993

[Metalloproteinase of Bacillus mesentericus, strain V-313].

A homogeneous metalloproteinase has been isolated with a 28% yield from the culture fluid of Bacillus mesentericus, strain B-313. The isolation procedure included chromatography on bacitracin-silochrome and gel filtration on Acrylex P-10 and Sephadex G-75. The enzyme has a molecular mass of 41,000 Da; its N-terminal sequence, which appears as A-A-T-T-G-T-G-T-T-L-K-G-K-T-V-S-L-N-I, is identical with that of the B. amyloliquefaciens enzyme. Like other metalloproteinases, the enzyme is inhibited by o-phenanthroline and EDTA, has an activity maximum at 55 degrees C and pH 6.5-7.2, is stable at pH 7.0-9.5 and at temperature below 45 degrees C for several hours, and is irreversibly inactivated in acid media. As can be judged from the kcat/Km ratio dependence on pH, two ionogenic groups with pKa of 7.4 and 6.2 are involved in the catalytic act, presumably the imidazol group of histidine and the carboxylic group. Within synthetic peptides the enzyme hydrolyzes the bonds formed by the amino group of hydrophobic amino acids, mostly of leucine residues.

Amino Acid Sequence↗
PubMed1993

[Isolation and characteristics of Bacillus megaterium metalloproteinase].

Stepwise application of affinity chromatography on bacitracin-silochrome, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-15, a homogeneous metalloproteinase (M(r) = 35,000 Da) has been isolated from the cultural filtrate of B. megaterium strain 599. The amino acid composition and N-terminal sequence (20 amino acids) of the enzyme have been determined. The proteinase is not inhibited by diisopropyl-fluorophosphate, is inhibited by o-phenanthroline, EDTA, and Zn2+, and is activated by Co2+. The enzyme has a peak activity at 60-65 degrees C. The maximum of the enzymatic activity after hydrolysis of synthetic substrates is at pH 6.5-7.0. The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 C for several hours. In acid media the enzyme undergoes irreversible inactivation. The dependence of kcat/Km on pH points to the involvement of an ionogenic group with pKa 7.5 in the catalytic act, most probably of the imidazole group of histidine. The metalloproteinase hydrolyzes synthetic peptide substrates at the bonds formed by the amino groups of hydrophobic amino acids-Phe, Leu, Ile and Val.

Amino Acid Sequence↗
PubMed1993

[Renaturation of bacterial metalloproteinases].

After denaturation by phenol or acid ethanol bacterial metalloproteinases secreted by Bacillus thermoproteolyticus and Bacillus megaterium cells can be renatured by dissolution in 99.7% formic acid with subsequent dilution of the solution and its neutralization with an appropriate amount of an alkali. Renaturation is optimal at pH 9.0 in the presence of 30% glycerol as stabilizer, Ca2+ and Zn2+ ions needed for the formation of a native structure and reconstitution of the enzyme catalytic center. The active enzyme yield is 60-80%. Reactivated metalloproteinases retain their enzymatic properties, amino acid composition and molecular mass. Under these conditions autolysis of metalloproteinases does not significantly influence their renaturation.

Amino Acid Sequence↗
PubMed1993

[Co-precipitation of pepsin with products from the enzymatic synthesis of peptides as a factor limiting the effectiveness of the enzyme].

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.

Amino Acid Sequence↗
PubMed1992

Nucleophile specificity in alpha-chymotrypsin- and subtilisin-(Bacillus subtilis strain 72) catalyzed reactions.

Nucleophilic properties of amino-acid amides were studied systematically in acyl-transfer reactions catalyzed by alpha-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) using Mal-L-Ala-L-Ala-L-PheOMe as the acyl-group donor. In alpha-chymotrypsin-catalyzed reactions, the nucleophile reactivities increase in the following order: D-AlaNH2 < GlyNH2 < L-AlaNH2 < L-SerNH2 < L-ThrNH2 < L-HisNH2 < L-ValNH2 < L-LeuNH2 < L-TrpNH2 < L-MetNH2 < L-NvaNH2 < L-PheNH2 < L-IleNH2 < L-TyrNH2 < L-ArgNH2. In reactions catalyzed by subtilisin 72, the reactivities increase as follows: L-LeuNH2 < L-IleNH2 < L-ThrNH2 < L-ArgNH2 < L-TrpNH2 < L-NvaNH2 < L-ValNH2 < L-MetNH2 < L-AlaNH2 < L-SerNH2 < D-AlaNH2 < GlyNH2. In alpha-chymotrypsin-catalyzed reactions, hydrophobic interactions are entirely responsible for the differences between the reactivity of the nucleophiles for amides of all the amino-acids tested with the exception of D-AlaNH2, L-ArgNH2 and L-TyrNH2. In reactions catalyzed by subtilisin 72, amino-acid side-chain characteristics and the nucleophile reactivities are not related. The data obtained show the low selectivity of the S1' subsite of subtilisin 72 and high specificity of this subsite in alpha-chymotrypsin.

Amides↗
PubMed1992

Organic solvent changes the chymotrypsin specificity with respect to nucleophiles.

In alpha-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 alpha-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.

Amino Acid Sequence↗
PubMed1992

A serine proteinase of an archaebacterium, Halobacterium mediterranei. A homologue of eubacterial subtilisins.

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).

Amino Acid Sequence↗
PubMed1992

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

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

Amino Acid Sequence↗
PubMed1992

Primary structure of carboxypeptidase T: delineation of functionally relevant features in Zn-carboxypeptidase family.

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.

Amino Acid Sequence↗
PubMed1992

Pepsin as a catalyst of peptide synthesis. Enzyme co-precipitation with emerging peptide products.

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.

Amino Acid Sequence↗