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PubMed1975

Circular dichroism and gel filtration behavior of subtilisin enzymes in concentrated solutions of guanidine hydrochloride.

The circular dichroism of diisopropylphosphorylsubtilisins Novo and Carlsberg in both the near- and farultraviolet spectral regions is unaltered by concentrations of guanidine hydrochloride as high as 4 M at neutral pH. At concentrations of guanidine hydrochloride greater than 4 M slow irreversible time-dependent changes, apparently obeying second-order kinetics, are evident in both the near- and far-ultraviolet circular dichroism of these enzymes. Gel filtration studies of inactivated subtilisin enzymes reveal the circular dichroism changes to be accompained by the appearance of aggregated protein material. The changes in circular dichroism and the production of associated subtilisin species are sensitive to protein concentration, denaturant concentrations, and pH. The circular dichroism of active subtilisins Novo and Carlsberg in guanidine hydrochloride exhibits irreversible changes similar to those observed for the inactivated subtilisins. Aggregated protein material is also formed initially in the presence of guanidine hydrochloride, but is rapidly autolyzed to low molecular weight fragments.

Binding Sites↗
PubMed1977

Subtilisin modification of monodeamidated ribonuclease-A.

Limited proteolysis of RNAase-Aa(1) (monodeamidated ribonuclease-A) by subtilisin results in the formation of an active RNAase-S type of derivative, namely RNAase-Aa(1)S. RNAase-Aa(1)S was chromatographically distinct from RNAase-S, but exhibited very nearly the same enzymic activity, antigenic conformation and susceptibility to trypsin as did RNAase-S. Fractionation of RNAase-Aa(1)S by trichloroacetic acid yielded RNAase-Aa(1)S-protein and RNAase-Aa(1)S-peptide, both of which are inactive by themselves, but regenerate active RNAase-Aa(1)S' when mixed together. RNAase-Aa(1)S-peptide was identical with RNAase-S-peptide, whereas the protein part was distinct from that of RNAase-S-protein. Titration of RNAase-Aa(1)S-protein with S-peptide exhibited slight but noticeably weaker binding of the peptide to the deamidated S-protein as compared with that of native protein. Unlike the subtilisin digestion of RNAase-A, which gives nearly 100% conversion into RNAase-S, the digestion of RNAase-Aa(1) gives only a 50% conversion. The resistance of RNAase-Aa(1) to further subtilisin modification after 50% conversion is apparently due to the interaction of RNAase-Aa(1) with its subtilisin-modified product. RNAase-S was also found to undergo activity and structural changes in acidic solutions, similar to those of RNAase-A. The initial reaction product (RNAase-Sa(1)) isolated by chromatography was not homogeneous. Unlike the acid treatment of RNAase-A, which affected only the S-protein part, the acid treatment of RNAase-S affected both the S-protein and the S-peptide region of the molecule.

Amino Acids↗
PubMed1976

Isolation and some properties of a subtilisin inhibitor from barley.

An inhibitor affecting subtilisin [EC 3.4.21.14] was isolated from barley (Hordeum vulgare L cv. Kikaihadaka) by extraction with 1% sodium chloride, fractionation with ammonium sulfate, chromatography on CM- and DEAE-cellulose columns, and gel filtration on Sephadex G-100. The final preparation appeared to be homogeneous on the basis of polyacrylamide gel electrophoresis; the inhibitory activity against subtilisin was increased about 140-fold during purification. This inhibitor was protein having a molecular weight of about 20,000, and containing 177 amino acid residues. Both the amino- and carboxyl-terminal residues were alaine. The inhibitor inactivated subtilisin, probably for formation of an enzyme-inhibitor complex in a molar ratio of 1: 1, but had little or no effect on the activities of other enzymes tested. The dissociation constant of the subtilisin-inhibitor complex was 1.5 X 10(-10) M. The inhibitor appears to be distinct from the barley microbial proteinase isoinhibitors reported by Mikola and Suolinna, in respect of most of its physiochemical and inhibitory properties.

Hordeum↗
PubMed1978

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.

Amino Acid Sequence↗
PubMed1978

[Immunochemical study of subtilisins obtained from Bacillus subtilis A-50 and some of its mutants].

Physico-chemical parameters of subtilisins from the original Bacillus subtilis A-50 strain (proteolytic activity, electrophoretic mobility, molecular weight, reactions with specific inhibitors) were similar to those mentioned in the literature for the enzymes of other strains. Immunological experiment has shown, that Bacillus subtilis A-50 subtilisins with various electrophoretic mobility do not differ in their antigenic properties. Enzymes with high electrophoretic mobility from mutant strains were similar to I--III subtilisin fractions from Bac. subtilis A-50 in the antigenic characteristics. However, the antigenic heterogeneity was observed in I, II and III enzyme fractions of some mutant strains. Subtilisins studied appear to form the isoenzyme system.

Antigen-Antibody Reactions↗
PubMed1979

[Affinity chromatography of subtilisin on a sorbent with epsilon-aminocapronyl-alanyl-alanyl-D-leucylamide. Detection of a serine protease with unusual properties].

A biospecific sorbent obtained by attachment of epsilon-aminocapronyl-L-alanyl-L-alanyl-D-leucylamide to CNBr-activated Sepharose 4B has been used for affinity chromatography of various samples of subtilisin BPN', e.g. subtilisin A ("Serva"), Nagarse, A-50. Two active components were isolated from subtilisin A (Serva"), the major component corresponding to subtilisin BPN' and the minor component (SII) being a serine proteinase with low molecular weight (about 10000). The molecular weight and amino acid composition of SII as well as the kinetic parameters of its action on peptide substrates (p-nitroanilides of N-benzyloxycarbonyl-Gly-Gly-Leu, -Ala-Ala-Leu, -Gly-Gly-Phe, -Ala-Ala-Phe. The low molecular weight proteinase possesses a high affinity for the leucine residue in P1 position and alanine in P2 and P3 positions. The specificity of this proteinase differs from that of the main component.

Chromatography, Affinity↗
PubMed1978

Binding of m-nitrobenzeneboronic acid to the active site of subtilisin BPN.

m-Nitrobenzeneboronic acid as a possible transition-state analog for serine proteases was found to cause absorption spectral change from 250 nm 350 nm upon binding with subtilisin BPN' (EC 3.4.21.14) at pH 6.5. Similar difference spectral changes of m-nitrobenzeneboronic acid were also observed at alkaline pH or upon addition of N-methylimidazole at pH 6.5. A characteristic circular dichroism spectrum of m-nitrobenezeneboronic acid was induced upon binding with subtilisin BPN' not only at pH 6.5, but also at alkaline pH. Circular dichroism spectral titration confirmed the stoichiometry of 1 : 1 for the m-nitrobenzeneboronic acid - subtilisin complex. m-Nitrobenzeneboronic acid was shown to be useful as a reversible chromophoric probe for the catalytic site of serine proteases.

Binding Sites↗
PubMed1976

Specific fluorescent derivatives of macromolecules. A fluorescence study of some specifically modified derivatives of chymotrypsin, trypsin and subtilisin.

The 5-dimethylaminonaphthalene-1-sulfonyl group was specifically introduced into the active site region of the serine proteinases: alpha-chymotrypsin, trypsin and subtilisin Carlsberg by the method of affinity-labeling. The resulting fluorescent derivatives were studied by a variety of fluorescence techniques and the results were correlated with structural data available on these enzymes from X-ray analysis. As model compounds for the Dns-proteinases, the absorption and fluorescence properties of Dns-amide and Dns-ethyl ester were studied in ethanol/water and p-dioxane/water mixtures. The fluorescence emission transtion energies and quantum yields were related to four commonly employed solvent-polarity scales. Best correlations for different solvents were obatined with the empirical "Z" and "Y" scales. From inspection of the fluorescence emission transition energies of the Dns group in the Dns-proteinases and comparision with the model compound studies it was possible to assign "Z" values for the apparent microenvironment polarities of the Dns group in the Dns-proteinases. The apparent polarities of the microenvironments of the Dns group in Dns-Ser 195-chymotrypsin (Dns-chymotrypsin (I)); (Dns-Phe-CH2)-His 57-chymotrypsin; (Dns-Lys-CH2)-His 46-trypsin; and Dns-Ser 221--subtilisin Carlsberg (Dns-subtilisin (I)) are in the range of 89.5-92.5 on the "Z" scale. The apparent microenvironment polarity of the Dns group in Dns-Ser 183-trypsin (Dns-trypsin (I)) appears to be below 76.7 on the "Z" scale. The Dns group in Dns-chymotrypsin (I) and (Dns-Phe-CH2)-His 57-chymotrypsin appears to be rigidly bound as evaluated by fluorescence polarization studies. The effect of 2H2O on the fluorescence emission quantum yields of Dns-amide and Dns-ethyl ester was examined. In both cases the ratios of quantum yields in 2H2O:ethanol (8:2) to quantum yields in H2O:ethanol (8:2) was about 1.8. The 2H2O effect upon the fluorescence emission quantum yields of the Dns group has been used to investigate solvent accessibility of this chromophore in the Dns-proteinases. Acessibility studies using 2H2O are very promising and have some definite advantages over other existing methods. Energy transfer between the Trp residues and the bound Dns group was investigated in the Dns-proteinases. The mean transfer distance calculated from the observed transfer efficiencies are 18.1 A, 19.7 A and 18.4 A for (Dns-Phe-CH2)-His 57-chymotrypsin, Dns-chymotrypsin (I) and (Dns-Lys-CH2)-His 46-trypsin, respectivly. From models built using X-ray crystallographic coordinates for the protein atoms, the mean distance of separation between the Trp residues and the bound Dns group for the same set of conjugates ar 18.6 A, 17.5 A and 17.5 A, respectively. Considering the inherent difficulties in energy transfer studies, the results are in excellent agreement with the X-ray data.

Chymotrypsin↗
PubMed1975

A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.

Circular Dichroism↗
PubMed1977

Interaction of thiolsubtilisin with Streptomyces subtilisin inhibitor, SSI.

Subtilisin BPN' was chemically converted to thiolsubtilisin and the interaction of this modified enzyme with Streptomyces subtilisin inhibitor (SSI) was examined. SSI competitively inhibited the esterolytic activity of thiolsubtilisin toward p-nitrophenyl acetate with a K1 value of 1.3 X 10(-5) M at pH 7.5 Spectrophotometric analysis of the interaction between SSI and the modified enzyme yielded a Kd value of 4 X 10(-5) M at pH 9.7. These values are about 10(5)-fold greater than the Kd value (less than 10(-9) M at pH 7.5) for the native enzyme. This indicates that the small change in the active site structure of subtilisin (Ser221 to Cys221) leads to a considerable decrease in the binding affinity (by about 6-7 kcal/mol) to SSI.

Acetates↗
PubMed1978

[Effect of Bacillus subtilis A-50 "streptomycin" mutations on the formation of molecular forms of subtilisin, an extracellular alkaline proteinase].

The patterns of subtilisin molecular forms of streptomycin-resistant (Strr) and streptomycin-dependent (Strd) mutants of Bacillus subtilis A-50, as well as the revertants of Strd to streptomycin-independence (Str1) were studied. Strr mutants had different quantitative pattern of the same subtilisin molecular forms as compared with the initial strain A-50 (the forms with Rf 0.08, 0.16 and 0.3). In comparison with the initial strain A-50, Strd mutants and Str1 revertants revealed three additional forms of the active enzyme with Rf 0.02, 0.5 and 0.7 and the molecular weights less than 35,000, 28,000 and 20,000 respectively. It was suggested that the rate and character of the enzyme secretion of the degree of its post-translational modifications might result in the different pattern of subtilisin molecular forms produced by these streptomycin mutants.

Bacillus subtilis↗
PubMed1975

Some properties of a protease (subtilisin BPN') immobilized to porous glass.

Subtilisin BPN' was immobilized to porous glass via isothiocyanate coupling. The pH optimum of the enzyme was shifted to the alkaline side on binding. This effect was more pronounced with ethyl lactate than with N-tosyl arginine methyl ester (TAME). Presumably, the shift is a reflection of the negative charge on the surface of the glass. The Michaelis constant and Vmax of soluble subtilisin BPN' with TAME were two and one orders of magnitude, respectively, lower than with ethyl lactate. Vmax, calculated per g of active enzyme, with TAME as the substrate was not affected by immobilization, while Vmax with ethyl lactate decreased greater than tenfold. The apparent KM decreased on immobilization with ethyl lactate as substrate and increased with TAME. Results are explained in terms of diffusional resistance and a possible attraction of ethyl lactate to the glass surface. Active site titration indicated that about 25% of the immobilized enzyme was active.

Glass↗
PubMed1976

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.

Bacterial Proteins↗
PubMed1978

The development of a fluorometric method for the assay of subtilisins.

An ultra-sensitive fluorometric method has been developed for quantitating subtilisins in aqueous solution. Using detergent solutions as blanks, the analytical sensitivity was estimated to be 0.002 microgram of 100% active enzyme per milliliter. The required sampling time for quantitating subtilisins in air at their TLV was estimated to be approximately two minutes.

Caseins↗
PubMed1977

Crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor, at 2.3 angstrom resolution.

The crystal structure of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor which strongly inhibits bacterial alkaline proteinases, was determined at 2.3 angstrom resolution. The subunit (molecular weight, 11,485) of this dimeric molecule has a unique fold of polypeptide chain with a five-fold anti-parallel beta-sheet structure (about 21% of the 113 amino acid residues) and two small segments of alpha-helices (about 16%). The region around the apparent reactive site, Met(73)-Val(74), is held tight by a combination of various structural features. The conformation of this region seems to have close similarity to that found in substrate analogues of low molecular weight bound to subtilisin BPN'.

Crystallography↗
PubMed1979

Interactions of BPN' and Carlsberg subtilisins with peptides containing aromatic amino acids at the C-terminus. Specific rate enhancement due to the secondary enzyme-substrate interaction.

The interaction between BPN' or Carlsberg subtilisins and peptides of the type Ac-Glyn-X-OMe (n = 0, 1, 2, 3), where X denotes one of five different aromatic amino acids, was investigated to elucidate the effect of the secondary interaction on catalysis in relation to the nature of the X residue. The increase in interaction upon elongation of the chain was accompanied by a large increase in kcat but with no marked change in Km in all the series of sensitive substrates. The peptides containing 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan, however, acted as competitive inhibitors and exhibited an invariant dissociation constant in spite of the different chain lengths. These observations suggest that the secondary enzyme-substrate interaction induces a conformational change in the active site of the enzyme or in the substrate in such a way as to lower the activation energy and to form a stabilized transient complex. In this respect, BPN' and Carlsberg subtilisins are similar to porcine pepsin and Streptomyces griseus protease 1 rather than to alpha-chymotrypsin.

Calorimetry↗
PubMed1979

A method for designing peptide substrates for proteases. Tripeptidyl-p-nitroanilide substrates for subtilisin Carlsberg.

1. The kinetic parameters of 25 peptidyl-p-nitroanilide substrates were investigated with subtilisin Carlsberg as model enzyme. 2. For a series of 12 substrates, the contribution of various side chains to the affinity constant was computed by regression analysis. From these contributions the sequence of a new and better substrate, N-benzyloxycarbonyl-arginyl-norleucyl-norleucyl-p-nitroanilide (Z-Arg-Nle-Nle-Nan) was predicted. The compound was synthesized and assayed. Its calculated 1/Km value, 43.5 mM-1, was in a good agreement with the value of 40.0 mM-1 that was determined experimentally. 3. On expanding the series to 19 substrates, it was found that the productivity of enzyme-substrate binding is influenced primarily by those subsites which have a significantly greater contribution to the affinity constants than others. 4. The additivity principle applied reasonably well for the contribution of individual side chains to the kinetic parameters. This fact suggests that regression analysis can be used for the prediction of the amino acid sequence of better substrates than those already tested, probably not only for subtilisin but also for other proteolytic enzymes.

Kinetics↗
PubMed1975

Use of N-benzoyl-L-tyrosine thiobenzyl ester as a protease substrate. Hydrolysis by alpha-chymotrypsin and subtilisin BPN.

In the course of searching for specific chromogenic substrates which might be useful in screening for protease-deficient mutants of Bacillus subtilis, we have developed a method for the synthesis of N-benzoyl-L-tyrosine thiobenzyl ester (BzTyrSBzl) in good yield. Spontaneous base hydrolysis of this thiol ester is low, but several serine proteases hydrolyze it readily. Spectrophotometric measurement of the hydrolysis of the ester in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) provides a continuous assay for chymotrypsin as sensitive as any assay reported in the literature. Serine proteases which hydrolyze this substrate may be detected in polyacrylamide disc gels by incubation in the presence of nitro blue tetrazolium. Apparent Km values of 0.02 and 7 mM and kcat values of 37 S-1 and 126 S-1 were observed for the hydrolysis of BzTyrSBzl by alpha-chymotrypsin and subtilisin BPN', respectively. Additionally, 5 mM indole was observed to behave as a strict competitive inhibitor of the alpha-chymotrypsin-catalyzed hydrolysis of BzTyrSBzl but was observed to increase the maximal rate of hydrolysis of p-nitrophenyl acetate by alpha-chymotrypsin by 30%, as previously described. These data, the published data of other workers, and results from studies with molecular models of trypsin and subtilisin BPN' are used as the basis for describing more fully a secondary hydrophobic binding pocket on alpha-chymotrypsin. The pocket is immediately adjacent to the active site serine and is tentatively suggested to be composed of 4 aliphatic side chain residues and 2 glycine residues.

Benzyl Compounds↗