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PubMed2000

The excluding effects of sucrose on a protein chemical degradation pathway: methionine oxidation in subtilisin.

The conformational stabilization of proteins by sucrose has been previously attributed to a preferential exclusion mechanism. The present study links this mechanism to stability against a chemical degradation pathway for subtilisin. Oxidation of a methionine residue adjacent to the active site to the sulfoxide form compromises subtilisin's enzymatic activity. In the presence of hydrogen peroxide and borate buffer, a borate-hydrogen peroxide complex binds to subtilisin's active site prior to the formation of methionine sulfoxide. Sucrose decreases the oxidation rate by limiting the accessibility of the complex to the methionine at the partially buried active site. The stabilization mechanism of sucrose is based on shifting the equilibrium of transiently expanding native conformations of subtilisin to favor the most compact states. Enzymatic parameter determination (kcat, KM) and hydrogen-deuterium exchange measurements confirm the limited conformational mobility of the enzyme in the presence of sucrose. Further support for limited mobility as the cause of oxidation inhibition by sucrose comes from the findings that neither viscosity nor possible interactions of sucrose with hydrogen peroxide, hydroxyl radicals, or borate can adequately explain the inhibition. The volume exclusion of sucrose from subtilisin is used to estimate the extent by which the native state of subtilisin must expand in solution to allow oxidation. The surface area of the oxidation-competent state is ca. 3.9% greater than that of the native state.

Borates↗
PubMed1988

Stability of aprA-subtilisin in sodium dodecyl sulfate.

The effect of sodium dodecyl sulfate (SDS) on the structure and activity of aprA-subtilisin, a secreted bacterial serine protease which is 85% homologous to subtilisin BPN', was examined. The addition of SDS resulted in the slow conversion of the subtilisin from the intact protein to the completely unfolded form of the enzyme. No intermediates between these two populations were detected. This conversion was accompanied by decreased activity, disruption of tertiary structure, a change in the mobility of the protein when subjected to SDS-polyacrylamide gel electrophoresis, and an increase in the apparent Stokes radius of the protein. After 2 h in 1% SDS at 20 degrees C, 25% of the subtilisin was still intact and active. The amount of protein existing in the unfolded form was increased by increasing the length of time in SDS, by increasing the concentration of SDS, and by increasing the temperature of the subtilisin-SDS solution. Analysis of the dependence of the rate of unfolding on SDS concentration indicated that one SDS micelle can destroy two protein molecules. The activation energy for the SDS-induced denaturation of aprA-subtilisin was 20 kcal mol-1, indicating that unfolding of the protein could be the rate-limiting step.

Bacillus subtilis↗
PubMed1992

Subtilisin cleavage of tubulin heterodimers and polymers.

Native pig brain tubulin in heterodimer or polymer form was subjected to limited proteolysis by subtilisin, which is known to cleave at accessible sites within the last 50 amino acids of the highly variable carboxyl-termini of the alpha and beta subunits. Heterodimeric tubulin or tubulin polymerized in the presence of 4 M glycerol or taxol was used in these experiments. Digested tubulin was purified by cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography in the absence or presence of nonionic detergent; however, smaller cleaved products of about 34,000 to 40,000 MW remained associated with the major cleaved subunits, alpha' and beta', under all purification conditions. In order to determine the effect of subtilisin cleavage on tubulin heterogeneity, purified native or subtilisin-cleaved tubulin was subjected to isoelectric focusing, followed by SDS-PAGE. The total number of isotypes was reduced from 17-22 for native alpha,beta tubulin to 7-9 for subtilisin-cleaved alpha',beta' tubulin. When tubulin heterodimers were cleaved, a single major beta' isotype was evident; however, when tubulin polymerized in 4 M glycerol was cleaved, two major beta' isotypes were found. Monoclonal antibodies that recognize a beta carboxyl-terminal peptide, residues 410-430, reacted with both major beta' isotypes, indicating that subtilisin cleavage occurred within the last 20 of the 450 amino acids. In order to establish whether this difference was in fact associated with polymer or heterodimer forms of tubulin, digestion was carried out in the presence of taxol, which stabilizes tubulin polymers. A single major beta' isotype different from the cleaved heterodimer, but coincident with one of the bands of the cleaved glycerol-induced polymers, was found when taxol-treated tubulin was digested. This result suggests the presence of more than one subtilisin site in the beta subunit, near residues 430-435, with different accessibility to the enzyme in the heterodimer and polymer form.

Animals↗
PubMed2004

The role of conformational flexibility of enzymes in the discrimination between amino acid and ester substrates for the subtilisin-catalyzed reaction in organic solvents.

To investigate how the conformational flexibility of subtilisin affects its ability to discriminate between enantiomeric amino acid and ester substrates for the subtilisin-catalyzed reaction in an organic solvent, the flexibility around the active site and the surface of subtilisin was estimated from the mobility of a spin label bound to subtilisin by ESR spectroscopy. Many studies on enzyme flexibility focus on the active site. Both the surface and active site flexibility play an important role in the enantioselectivity enhancement of the enzyme-catalyzed reaction. It was found, however, that the different behavior observed for the enantioselectivity between the amino acid and ester substrates could be correlated with the flexibility around the surface rather than the flexibility at the active site of subtilisin. In other words, for the ester substrates, the greater flexibility around the surface of subtilisin induced by a conformational change resulting from the presence of an additive such as DMSO is essential for the enantioselectivity enhancement. This model is also supported by the Michaelis-Menten kinetic parameters for each enantiomeric substrate. Our findings provide insight into the enantioselectivity enhancement for the resolution of enantiomers for enzyme-catalyzed reactions in organic solvents.

Amino Acids↗
PubMed1994

Variants of subtilisin BPN' with altered specificity profiles.

A strategy for increasing the size of the S4 binding pocket was used to improve the specificity of subtilisin BPN' toward substrates with large hydrophobic P4 side chains. This approach involves single and double amino acid replacements at positions 104, 107, and 126. Previously, alteration of I107 to glycine has been found to increase the specificity of subtilisin toward leucine, isoleucine, and phenylalanine as P4 residues by up to 214-fold. Replacement of Y104 by alanine also yields a similar improvement in specificity. However, this subtilisin variant favors isoleucine and phenylalanine over leucine. When L126 was replaced by valine, alanine, and glycine, respectively, only the L126A subtilisin variant, which possesses a 28-fold-increased catalytic efficiency for isoleucine compared with all other substrates tested, showed a significantly improved specificity profile. As inferred from the double-mutant enzymes I107G/L126V, I107G/L126A, and I107G/Y104A, none of the effects of the single amino acid replacements on the kinetic parameters are additive. The I107G/L126V mutant subtilisin has the largest improvement in P4 substrate specificity reported so far: kcat/KM is increased 340-fold for leucine compared to alanine. By contrast, the specificity profile of the I107G/Y104A mutant enzyme is impaired in comparison with that of the corresponding single mutants. Therefore, the design of high-specificity subtilisin variants through the combination of single amino acid replacements in the S4 pocket appears to be nontrivial due to the interference of the introduced structural changes.

Amino Acid Sequence↗
PubMed1988

Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis.

A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKas for the six histidines in this enzyme. The pKas of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.

Bacillus↗
PubMed2001

Cation-dependent stability of subtilisin.

Subtilisin BPN' contains two cation binding sites. One specifically binds calcium (site A), and the other can bind both divalent and monovalvent metals (site B). By binding at specific sites in the tertiary structure of subtilisin, cations contribute their binding energy to the stability of the native state and increase the activation energy of unfolding. Deconvoluting the influence of binding sites A and B on the inactivation rate of subtilisin is complicated, however. This paper examines the stabilizing effects of cation binding at site B by using a mutant of subtilisin BPN' which lacks calcium site A. Using this mutant, we show that calcium binding at site B has relatively little effect on stability in the presence of moderate concentrations of monovalent cations. At [NaCl] =100 mM, site B is >or=98% occupied with sodium, and therefore its net occupancy with a cation varies little as subtilisin is titrated with calcium. Exchanging sodium for calcium results in a 5-fold decrease in the rate of inactivation. In contrast, because of the high selectivity of site A for calcium, its occupancy changes dramatically as calcium concentration is varied, and consequently the inactivation rate of subtilisin decreases approximately 200-fold as site A becomes saturated with calcium, irrespective of the concentration of monovalent cations.

Bacillus↗
PubMed2004

Engineering subtilisin into a fluoride-triggered processing protease useful for one-step protein purification.

Subtilisin was engineered into a highly specific, processing protease, and the subtilisin prodomain was coengineered into an optimized recognition sequence. This involved five steps. First, a robust subtilisin mutant was created, which could tolerate the subsequent mutations needed for high specificity. Second, the substrate binding pocket was mutated to increase its sequence selectivity. Third, the subtilisin prodomain was engineered to direct cleavage to the junction of any protein fused to it. Fourth, the active site of subtilisin was engineered to kinetically isolate binding and cleavage reactions. Finally, specific anions were identified to trigger the processing reaction, with fluoride ions being particularly useful. The ability to isolate the binding and processing steps with a triggering mechanism created a protease with a virtual on-off switch. This allowed column-immobilized processing subtilisin to be used as both the affinity ligand and processing protease for one-step purification of proteins. Fusion proteins tagged with the engineered prodomain can be bound to the column and washed free of contaminants. Cleavage can be triggered by the addition of fluoride to release the pure target protein. The column is then regenerated by stripping off the tightly bound prodomain at pH 2.1. Ten proteins have been purified to date by this method.

Amino Acid Substitution↗
PubMed2004

Binding, proteolytic, and crystallographic analyses of mutations at the protease-inhibitor interface of the subtilisin BPN'/chymotrypsin inhibitor 2 complex.

A series of mutants of chymotrypsin inhibitor 2 (CI2), at residues that interact with the inhibited enzyme subtilisin BPN', were studied to determine the relative importance of intermolecular contacts on either side of the scissile bond. Mutants were tested for inhibition of subtilisin, rates of hydrolysis by subtilisin, and ability to acylate subtilisin. Additionally, crystal structures of the mutant CI2 complexes with subtilisin were obtained. Ordered water molecules were found to play an important role in inhibitor recognition, and features of the crystal structures, in combination with biochemical data, support a transition-state stabilization role for the P(1) residue in subtilisin catalysis. Consistent with the proposed mechanism of inhibition, in which rapid acylation is followed by religation, leaving-group contacts with the enzyme were found to be more critical determinants of inhibition than acylating-group contacts in the mutants studied here.

Acylation↗
PubMed1999

Electric fields in active sites: substrate switching from null to strong fields in thiol- and selenol-subtilisins.

Although known to be important factors in promoting catalysis, electric field effects in enzyme active sites are difficult to characterize from an experimental standpoint. Among optical probes of electric fields, Raman spectroscopy has the advantage of being able to distinguish electronic ground-state and excited-state effects. Earlier Raman studies on acyl derivatives of cysteine proteases [Doran, J. D., and Carey, P. R. (1996) Biochemistry 35, 12495-502], where the acyl group has extensive pi-electron conjugation, showed that electric field effects in the active site manifest themselves by polarizing the pi-electrons of the acyl group. Polarization gives rise to large shifts in certain Raman bands, e.g. , the C=C stretching band of the alpha,beta-unsaturated acyl group, and a large red shift in the absorption maximum. It was postulated that a major source of polarization is the alpha-helix dipole that originates from the alpha-helix terminating at the active-site cysteine of the cysteine protease family. In contrast, using the acyl group 5-methylthiophene acryloyl (5-MTA) as an active-site Raman probe, acyl enzymes of thiol- or selenol-subtilisin exhibit no polarization even though the acylating amino acid is at the terminus of an alpha-helix. Quantum mechanical calculations on 5-MTA ethyl thiol and selenol ethyl esters allowed us to identify the conformational states of these molecules along with their corresponding vibrational signatures. The Raman spectra of 5-MTA thiol and selenol subtilisins both showed that the acyl group binds in a single conformation in the active site that is s-trans about the =C-C=O single bond. Moreover, the positions of the C=C stretching bands show that the acyl group is not experiencing polarization. However, the release of steric constraints in the active site by mutagenesis, by creating the N155G form of selenol-subtilisin and the P225A form of thiol-subtilisin, results in the appearance of a second conformer in the active sites that is s-cis about the =C-C=O bond. The Raman signature of this second conformer indicates that it is strongly polarized with a permanent dipole being set up through the acyl group's pi-electron chain. Molecular modeling for 5-MTA in the active sites of selenol-subtilisin and N155G selenol-subtilisin confirms the findings from Raman spectroscopic studies and identifies the active-site features that give rise to polarization. The determinants of polarization appear to be strong electron pull at the acyl carbonyl group by a combination of hydrogen bonds and the field at the N-terminus of the alpha-helix and electron push from a negatively charged group placed at the opposite end of the chromophore.

Acylation↗
PubMed1993

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.

Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment--high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtilisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60-->Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures--it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.

Amino Acid Sequence↗
PubMed1999

Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin.

RNA ligands (RNA aptamers) to a protease subtilisin were selected from pools of random RNA by SELEX (systematic evolution of ligands by exponential enrichment) and by use of a subtilisin-immobilized Sepharose column. After eight rounds of selection, RNA aptamers were isolated by cloning to a plasmid vector. We characterized one of the selected RNA molecules. This RNA aptamer displayed specific inhibition toward the subtilisin activity, even when the assay for subtilisin was performed using the chromogenic small peptide as substrate, and almost no inhibitory activity toward trypsin and chymotrypsin, although these enzymes are serine proteases similar to subtilisin. These findings indicate that this RNA can differentially recognize the surfaces of similar proteases. Kinetic analysis of the RNA aptamer revealed that the inhibition constant (Ki) toward subtilisin was 2.5 microM.

Animals↗
PubMed2000

Engineering subtilisin E for enhanced stability and activity in polar organic solvents.

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).

Bacillus subtilis↗
PubMed1992

The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis.

Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.

Amino Acid Sequence↗
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↗
PubMed1990

Effect of inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI.

The protein Streptomyces subtilisin inhibitor, SSI, efficiently inhibits a bacterial serine protease, subtilisin BPN'. We recently demonstrated that functional change in SSI was possible simply by replacing the amino acid residue at the reactive P1 site (methionine 73) of SSI. The present paper reports the additional effect of replacing methionine 70 at the P4 site of SSI (Lys73) on inhibitory activity toward two types of serine proteases, trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversion of methionine 70 at the P4 site of SSI(Lys73) to glycine or alanine resulted in increased inhibitory activity toward trypsin and lysyl endopeptidase, while replacement with phenylalanine weakened the inhibitory activity toward trypsin. This suggests that steric hindrance at the P4 site of SSI(Lys73) is an obstacle for its binding with trypsin. In contrast, the same P4 replacements had hardly any effect on inhibitory activity toward subtilisin BPN'. Thus the subsite structure of subtilisin BPN' is tolerant to these replacements. This contrast in the effect of P4 substitution might be due to the differences in the S4 subsite structures between the trypsin-like and the subtilisin-like proteases. These findings demonstrate the importance of considering structural complementarity, not only at the main reactive site but also at subsites of a protease, when designing stronger inhibitors.

Bacterial Proteins↗
PubMed1992

Spectroscopic studies on proteinase K and subtilisin DY. Relation to X-ray models.

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65 degrees and 48 degrees, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.

Circular Dichroism↗
PubMed2005

Gene expression and characteristics of a novel fibrinolytic enzyme (subtilisin DFE) in Escherichia coli.

AIMS: The objective of this study is to actively express a novel fibrinolytic enzyme, subtilisin DFE (douchi fibrinolytic enzyme), in Escherichia coli. METHODS AND RESULTS: The DNA fragments encoding pro-subtilisin DFE was amplified and cloned into the vector pET32a to obtain N-terminal Trx fusion expression plasmid. The recombinant subtilisin DFE was successfully expressed and processed in the soluble fraction of E. coli BL21(DE3) in a similar fashion as the endogenous one of Bacillus amyloliquefaciens DC-4, resulting in an active enzyme. Moreover, active enzyme can also be refolded from inclusion body. CONCLUSIONS: Active subtilisin DFE can be expressed and processed in E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidences that subtilisin DFE can be actively expressed in E. coli and the pro-peptide is essential for guiding the proper folding into the active conformation. As such, large quantities of recombinant subtilisin DFE can be produced for pharmacological and clinical research.

Bacillus↗