Valentin M. Stepanov

Comparative thermodynamic study of pepsinogen and pepsin structure

Abstract Pepsinogen, pepsin and its C-terminal fragment have been studied thermodynamically in solution by a scanning microcalorimetric method at various pH and salt content values. It has been shown that: 1. (1) thermal denaturation of pepsinogen is a highly reversible process that takes place within a narrow temperature range depending upon the prevalent conditions, but under no condition does it correspond to a two-state transition. This process can be approximated by two quasi-independent transitions, indicating that the pepsinogen molecule consists of two slightly interacting, co-operative structural blocks of different size. 2. (2) Thermal denaturation of pepsin is a complex process that proceeds in two distinct stages occurring at different temperatures, only the second being completely reversible. These stages correspond to separate meltings of two independent parts of the molecule, these being the N-terminal lobe and the more stable C-terminal lobe. Neither of these stages represents a two-state transition. Analysis of these transitions shows that both parts of the pepsin molecule consist of two quasi-independent, co-operative units. 3. (3) All four co-operative units of pepsin have a compact structure with a welldeveloped hydrophobic core, and therefore these units should be regarded as structural domains of the molecule. Consequently, each lobe of the pepsin molecule represents a structural block consisting of two domains. In the presence of pepstatin, the two domains in the N terminal lobe co-operate to form a single system. 4. (4) In pepsinogen, the above-mentioned blocks are much less independent than the co operative units in pepsin, and it is likely that each of them consists of two merged domains. Therefore, release of the 44-residue N-terminal polypeptide upon activation of pepsinogen leads to a loosening of the domain structure and to an increase of interdomain motility of the molecule.

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide--a chromogenic substrate for thiol proteinase assay.

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.

Phenylboronic acid as a ligand for biospecific chromatography of serine proteinases

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.

Macluralisin - a serine proteinase from fruits of Maclura pomifera (Raf.) Schneid.

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.

Organic solvent changes the chymotrypsin specificity with respect to nucleophiles

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.

Glutamyl endopeptidase of Bacillus intermedius, strain 3‐19

© 1997 Federation of European Biochemical Societies.

A new subfamily of microbial serine proteinases? Structural similarities of Bacillus thuringiensis and Thermoactinomyces vulgaris extracellular serine proteinases

Abstract Extracellular serine proteinases produced by two taxonomically remote microorganisms - B. thuringiensis and T. vulgaris were shown to share common structural and functional features. Both enzymes contain cysteine residue apparently essential for their activity. Their N-terminal sequences are clearly homologous (10 coinciding residues among 14 compared), whereas only marginal extent of homology could be found when the N-terminal sequences of these enzymes were aligned with those of subtilisins. It is suggested that within the family of evolutionary related bacterial serine proteinases exists a subfamily of SH-containing serine proteinases.

Bacillus intermedius Glutamyl Endopeptidase. Molecular Cloning and Nucleotide Sequence of the Structural Gene

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.

Nucleotide sequence of a novel δ‐endotoxin gene cryIg of Bacillus thuringiensis ssp. galleriae

A gene cryIg coding for entomocidal protein δ‐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 δ‐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 δ‐endotoxin follows the same type of polypeptide chain folding as other Cryl proteins, whereas peculiarities of primary structure help to explain its unique specificity.

Affinity chromatography of proteolytic enzymes on silica-based biospecific sorbents.

New biospecific sorbents for affinity chromatography of proteolytic enzymes were prepared by the attachment of the cyclopeptide antibiotics bacitracin, bacilliquin or gramicidin S to aminosilochrom via a reaction with p-benzoquinone. The content of the cyclopeptide ligands within the sorbents varied from 2 to 46 mumol/g. The sorbents prepared by this reaction were successfully applied in the purification of the carboxylic proteinases produced by fungi, Russula decolorans (a basidiomycete) and Trichoderma lignorum, as well as crude pepsin. Serine proteinases from Thermoactinomyces vulgaris, Trichoderma koningii, Trichoderma lignorum and bacilli (subtilisins) were also submitted to chromatography on these materials. The yields of purified enzymes approached quantitative levels, sometimes being higher as a result of elimination of inhibitors. An important advantage of these sorbents is their stability against the enzymes degrading the carbohydrate matrixes of affinity sorbents synthesized on the basis of agarose, dextran or cellulose derivatives.