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At least 19 recordsLinked to original sources
PubMed1978

The determination of molecular weights of Streptomyces subtilisin inhibitor and the complex of Streptomyces subtilisin inhibitor and subtilisin BPN' by sedimentation equilibrium.

The molecular weight of Streptomyces subtilisin inhibitor (SSI), a protein proteinase inhibitor, and that of the complex of SSI and subtilisin BPN' [EC 3.4.21.14] were determined by a sedimentation equilibrium method in 25 mM phosphate buffer, at pH 7.0, ionic strength 0.1 M (NaCl), 25.0 degrees C. The molecular weight of SSI was found to be 23,000 over a wide concentration range, 0.01-10 mg/ml, the range used for inhibitory, spectrophotometric, and kinetic measurements. Based on the amino acid sequence, the molecular weight of SSI has been calculated to be 11,500 (Ikenaka, T., et al. (1974) J. Biochem. 76, 1191-1209); therefore, the molecular weight of 23,000 obtained above suggests that SSI is in a dimeric form under usual conditions in the concentration range of 5 X 10(-7)-5 X 10(-4) M. The molecular weight of the subtilisin BPN'-SSI complex was determined to be 78,000 in the concentration range of 0.03-5.0 mg/ml by sedimentation equilibrium of the crystallized preparation and by that of a mixture of subtilisin BPN' and SSI treated as a multicomponent-polydisperse system. The molecular weight obtained here, combined with the results of binding stoichiometry (Inouye, K., et al. (1977) J. Biochem. 82, 961-967) that showed that one mol of SSI (molecular weight, 11,500) and one mol of the enzyme (molecular weight, 27,500) are tightly bound (Kd less than 1 nM), demonstrate that one mol of dimeric SSI binds two mol of the enzyme to form a stable complex, E2I2.

Bacterial Proteins↗
PubMed1979

[Subtilisin 72: a serine protease from Bac. subtilis strain 72 - an enzyme similar to subtilisin Carlsberg].

Subtilisin 72, a serine proteinase secreted by Bac. subtilis strain 72 was purified by covalent chromatography on Sepharose sorbent containing p-(omega-aminomethyl)phenylboronic acid as a ligand. The homogeneity of subtilisin 72 was confirmed by isoelectrofocusing in a thin layer of polyacrylamide gel (pl 8.6). The amino acid composition of this enzyme is different from that of other subtilisins, e. g. subtilisin Carlsberg. The N = terminal amino acid sequence of subtilisin 72 traced up to the 35th residue turned to be the same as that of subtilisin Carlsberg with the exception of the 21st (Tyr) and the 30th (Ile) residues. This very pronounced extent of homology shows that subtilisin 72 is very similar although not identical to subtilisin Carlsberg.

Amino Acid Sequence↗
PubMed1979

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.

Amino Acids↗
PubMed1978

Direct fluorometric determination of a dissociation constant as low as 10(-10) M for the subtilisin BPN'--protein proteinase inhibitor (Streptomyces subtilisin inhibitor) complex by a single photon counting technique.

It was found that an increase in fluorescence intensity at 340 nm is observed on the binding of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' in the pH range 6--10. The dissociation constant, Ki, of the enzyme-inhibitor complex was determined as a function of pH and temperature by direct fluorometric titration utilizing the single photon counting technique in the protein concentration range of 10(-9) M. Ki values as low as 10(-10) M could be obtained with reasonable accuracy by this high-sensitivity detection method. From the temperature dependence of Ki, it was found that the binding is endothermic, and is entirely "entropy-driven" in nature. The effect of pH on Ki suggested the participation of an ionizable group with pKapp = 8.5 in the binding.

Bacterial Proteins↗
PubMed1976

Preparation and enzymatic properties of subtilisin Novo chemically attached to soluble DEAE-dextran and insoluble DEAE-sephadex.

Analogous soluble and insoluble derivatives of subtilisin Novo (EC 3.4.21.14) were prepared by coupling the enzyme to CNBr-activated DEAE-dextran and DEAE-Sephadex, respectively. The DEAE-dextran-subtilisin displayed pH optima and Km values for ester hydrolysis similar to subtilisin, whereas the pH versus activity profiles obtained with DEAE-Sephadex-subtilisin were shifter towards the alkaline pH region and the Km values were increased. Compared with subtilisin, DEAE-dextran-subtilisin showed a 40-65% reduction of kcat for hydrolysis of N-acetyl-L-tyrosine ethyl ester, p-tosyl-L-arginine methyl ester and benzyloxycarbonyl-glycyl-L-tyrosinamide and its maximum velocities for digestion of casein and clupein also amounted to 40-60% of the subtilisin values. With Deae-sephadex-subtilisin, in contrast, the maximum velocity of hydrolysis decreased to a greater extent for polypeptide substrates compared to ester substrates. The present results indicate that the chemical nature of a support can effect intrinsic properties of a matrix-bound enzyme in addition to the steric and diffusional effects usually observed with polymer-attached enzymes.

Caseins↗
PubMed1979

Comparison of the kinetic specificity of subtilisin and thiolsubtilisin toward n-alkyl p-nitrophenyl esters.

The p-nitrophenyl esters of straight-chain fatty acids were used as substrates of the enzyme subtilisin Novo (EC 3.4.4.16) and its chemically produced artificial enzyme thiolsubtilisin. Subtilisin and thiolsubtilisin pH--activity profiles were determined, and kinetic effects of the active site O-S substitution were observed. Among the substrates tested, both enzymes show highest specificity with p-nitrophenyl butyrate. It was also found that subtilisin is more sensitive to changes in substrate chain length than is thiolsubtilisin. Second-order acylation rate constants (k2/Ks) are remarkably similar for both enzymes. However, thiolsubtilisin deacylation rate constants and Km values are lower than analogous subtilisin constants. While thiolsubtilisin deacylation rate constants give a pH profile identical with that of subtilisin, the pH profile of thiolsubtilisin acylation rate constants shows an active site pK value lowered from the subtilisin pK of 7.15 and exhibits an inflection point at pH 8.45, which is absent in subtilisin.

Binding Sites↗
PubMed2016

Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease.

Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD.

Bacteria↗
PubMed1977

[Effects of mutations in Bacillus subtilis genome decreasing the protease activity on the formation of subtilisin molecular forms].

Multiple molecular forms of subtilisin--extracellular serine protease produced by the wild strain Bac. subtilis A-50 and its mutant strains with the protease activity decreased two-fold and more were studied. Six molecular forms of subtilisin were found on the whole when 33 mutant strains have been investigated under the experimental conditions. It is essential that both the wild and each of mutant strains under study produced not more than three out of these six forms. Three molecular forms of subtilisin from the mutant strains are similar to those found in the wild strain A-50, and have the molecular weight, of 27 000-30 000. Three other forms of subtilisin were revealed only in the mutant strains, and had the molecular weight of about 20 000. Apparently there is only one structural gene for subtilisin in Bac. subtilis genome. The appearence of multiple molecular forms of subtilisin may be due to the post-translational modifications (limited proteolysis) of the initial type of enzyme, i.e. pre-subtilisin. Probably, that certain mulations not affecting the structural gene can significantly change the expression of such gene by varying of the degree of product modifications.

Bacillus subtilis↗
PubMed1978

Kinetic specificities of BPN' and Carlsberg subtilisins. Mapping the aromatic binding site.

The kinetic specificities of BPN' and Carlsberg subtilisins [EC 3.4.21.14] were examined with various nucleus-substituted derivatives of Nalpha-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of alpha-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two sutilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar trans-p-phenylazobenzoyl group at the OH-function, with the smallest Km value showed that these enzymes possess a more extended aromatic binding site than has so far been demonstrated. Ac-Phe(4-NO2)-OMe was remarkable in being hydrolyzed with a particularly large kcat value (5,500 +/- 700 s-1 at pH 7.8 for Carlsberg subtilisin). Ac-Phe(4-NO2)-OMe and Ac-Tyr-OMe were distinguished by Carlsberg subtilisin in terms of kcat but not by BPN' subtilisin, suggesting that the specificity site of the former is more sensitive to a small change in size of substituent than that of the latter. Ac-Trp(NCps)-OMe and Ac-Trp(NCps)-OH were bound to the enzyme's active site but in a competitive manner. A difference in the standard free energies of binding between the two enzymes may indicate that the hydrophobic cleft of Carlsberg subtilisin is somewhat deeper and/or narrower than that of BPN' subtilisin.

Bacillus↗
PubMed1977

[Multiple forms of Bacillus subtilis subtilisin and effects of mutations on the distribution of its molecular forms].

Using polyacrylamide-gel electrophoresis, isoelectric focusing and gel-filtration it was demonstrated that the auxotrophic mutant strains of Bac. subtilis A-50 and their prototrophic revertant strains produce multiple molecular forms of subtilisin. Three of them are the same as the corresponding molecular forms of subtilisin from the wild strain A-50. In different mutant strains the relative amounts of the main three forms varies considerably resulting in the absence of certain forms in several strains. There is the additional minor form of subtilisin possessing high electrophoretic mobility in four prototrophic revertant strains and one Arg--auxotrophic strain of Bac. subtilis A-50. It would be reasonable to suppose that different molecular forms of subtilisin derive from the product of its single structural gene as a result of post-translational modifications (limited proteolysis). This enzyme and probably most, if not all secretory proteins may be synthesised as larger precursors and then specifically modified in the bacterial cell membranes. Thus, certain mutations, without affecting the structural gene of this secretory protein -- subtilisin -- have pronounced effects on this structural gene expression, varying the degree of its product modification and the amount of resulting secretory molecular forms of subtilisin.

Bacillus subtilis↗
PubMed1977

Resolution of independently titrating spectral components in the ultraviolet circular dichroism of subtilisin enzymes by matrix rank analysis.

The ultraviolet circular dichroism of di-isopropylphophoryl-subtilisins Carlsberg and Novo (EC 3.4.21.14) has been examined as a function of pH. The CD of these enzymes below 260 nm is invariant over the pH interval 4 to 12, below or above which spectral changes occur suggesting a transition to a random coil form. Above pH 8 contributions due to the ionization of tyrosyl residues appear in the CD above 260 nm as bands shifted to longer wavelengths. Three independently titratable components, obtained by matrix rank analysis, account for the observed CD spectral changes above 260 nm of Dip-subtilisin Carlsberg in the pH interval 8 to 12. By contrast, two components were derived for the Novo enzyme. The identities of the matrix rank components were surmised from their apparent pKa values. One component of both subtilisin enzymes corresponds to the CD of the "buried" or irreversibly titratable tyrosyl residues of the enzyme. The other matrix rank components correspond to the CD of the "exposed" or freely ionizable tyrosyl residues. These residues are optically active only in the ionized state. Two types of "exposed" tyrosyl residues, arising because of differing sensitivity to the ionization of the "partially buried" or abnormally titrating tyrosyl residues, are evident in Dip-subtilisin Carlsberg. A pH-induced local conformational change in this enzyme is proposed to account for this behavior. The "partially buried" tyrosyl residues of both subtilisins appear to be devoid of optical activity in either the tyrosyl or tyrosylate form.

Circular Dichroism↗
PubMed1976

Inactivation of Streptomyces subtilisin inhibitory by chemical modifications.

1. The inhibitory activity of an alkaline protease inhibitor, (Streptomyces subtilisin inhibitor) towards subtilisin is found to decrease by photooxidation sensitized by methylene blue with a clear pH dependence, the midpoint of which is about 6.0. 2. Amino acid analyses of photooxidized Streptomyces subtilisin inhibitor indicate that one of the two histidyl residues and the three methionyl residues are destroyed, concomittant with the loss of inhibitory activity. 3. In accordance with this observation, one of the clearly resolved nuclear magnetic resonances from C2-protons of the two histidyl residues is selectively diminished. This histidyl residue, sensitive to photooxidation and giving a proton magnetic resonance peak at lower field, is assigned to His-106 from peptide analyses. 4. Independent modification of methionyl residues by a reaction with H2O2 or Cl2 also decreases the inhibitory activity of Streptomyces subtilisin inhibitor. 5. Modification of lysyl, tyrosyl and tryptophanyl residues by diazonium-1-H-tetrazole does not lead to the loss of the inhibitory activity. 6. The above results indicate that one or more methionyl residue(s) are essential to the inhibitory activity of Streptomyces subtilisin inhibitor, whereas lysyl, tyrosyl and tryptophanyl residues are not essential to the inhibitory activity. Modification of His-106 is also strongly related to the loss of activity, although its distinct participation in the inactivation mechanism has not been demonstrated.

Amino Acid Sequence↗
PubMed1976

Denaturation of subtilisin BPN' and its derivatives in aqueous guanidine hydrochloride solutions.

The denaturation of subtilisin BPN' (EC 3.4.21.14) in guanidine hydrochloride was studied in order to find possible reasons for the exceptional stability of this enzyme against the action of denaturing agents including guanidine hydrochloride. Chemically modified subtilisins, i.e., phenylmethanesulfonylsubtilisin and thio-subtilisin, were completely denatured in 2 M guanidine hydrochloride at pH 7 without autolysis but they were stable in 0.5 M guanidine hydrochloride for at least 60 h. On the other hand, once completely denatured, the subtilisins remained inactive and in highly unfolded conformations for 60 h or longer after transfer into 0.5 M guanidine solution at pH 7 or 9. No enzymatic activity was regained when the guanidine concentration was lowered to almost zero. We concluded from these and other results described in this paper that this enzyme was thermodynamically unstable in 2 M guanidine hydrochloride at 20 degrees C and at pH 7. We wish to point out the possibility that the denaturation of this enzyme could indeed be irreversible.

Guanidines↗
PubMed1977

Inhibition of subtilisin BPN' with peptide chloromethyl ketones.

The kinetics of the reaction of a series of peptide chloromethyl ketones with subtilisin BPN' (EC 3.4.21.14) were investigated in order to correlate the rates of reaction in solution with the number of interactions which are observed in the cystallographic model of the inhibited enzyme (Robertus, J.D., Alden, R.A., Birktoft, J.J., Powers, J.C. and Wilcox, P.E. (1972) Biochemistry 11, 2439--2449). The second-order rate constant kobs/[I], measured at pH 7.0 in 9% 1,2-dimethoxyethane at 30 degress C, varied by a factor of 5900 from the slowest (Z-TrpCH2Cl) to the fastest inhibitor (Ac-Phe-Gly-Ala-LeuCH2Cl). Inhibitors with a P1 leucine or phenylalanine residue are equally effective. The increased reactivity of inhibitors containing alanine as the P2 residue is the result of a favorable contact between the methyl side chain of the alanyl residue and the S2 subsite of subtilisin. This result correlates nicely with the previously observed "secondary specificity" of subtilisin for substrates with alanine as the P2 residue. Teetrapeptide and tripeptide chloromethyl ketone inhibitors have kobs/[I] values of over 100-fold greater than those of most dipeptide and amino acid chloromethyl ketones. This again agress with the crystallographic model since tripeptide and tetrapeptide inhibitors could form a beta-sheet structure involving three or four hydrogen bonds with the enzyme while the others would form fewer hydrogen bonds. The S4 subsite of subtilisin exhibits a distinct preference for aromatic groups and our four most reactive inhibitors (Ac-Phe-Gly-Aca-LeuCH2Cl, Z-Gly-PheCH2Cl, Z-Gly-Gly-LeuCH2Cl, and Boc-Ala-Gly-PheCH2Cl) have either an aromatic (or a large hydrophobic) group as the P4 residue. The results demonstrate that the solution reactivity of peptide chloromethyl ketones can be explained on the basis of the crystal structures of chloromethyl ketone-inhibited substilisin derivatives and that substrate hydrolysis rates can be used to design effective chloromethyl ketone inhibitors for serine proteases.

Amino Acid Chloromethyl Ketones↗
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↗