Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Subtilisin”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 631 records · Page 35Linked to original sources
PubMed1977

Quenching of the tyrosyl and tryptophyl fluorescence of subtilisins Carlsberg and Novo by iodide.

The tyrosyl and tryptophyl fluorescence of diisopropylphosphorylsubtilisins Carlsberg and Novo, respectively, is quenched efficiently by I- but is not significantly affected by Cs+. The I-quenching data were analyzed using a modified Stern-Volmer treatment (Lehrer, S.S. (1971), Biochemistry 10, 3254), yielding values for the effective fraction of accessible protein fluorescence of 90-95 and 88-92% for the tyrosyl and tryptophyl emission of diisopropyl-phosphorylsubtilising Carlsberg and Novo, respectively. Similar values were obtained pH 5 and 7. The effective collisional quenching constant depends on pH in a manner suggesting the participation of protein surface charge in the quenching mechanism. Significant singlet energy transfer (efficiency = 0.52) from tyrosyl to tryptophyl residues was inferred from the excitation spectra of diisopropylphosphorylsubtilisn Novo. The very low efficiency of energy transfer to Trp-113 in diisopropylphorphorylsubtilisin Carlsberg suggests that Trp-105 and Trp-241 are the acceptors of tyrosyl emission in the homologous Novo enzyme. The unusually low quantum yield of Trp-113 in diisopropylphosphorylsubtilisin Carlsberg together with the tryptophyl fluorescence quenching behavior of the Novo enzyme suggests that this residue is "buried" and in accessible to quenching in both enzymes. The tyrosyl quenching behavior of diisopropylphorphorylsubtilisin Carlsberg is consistent with the high degree of solvent exposure of aromatic residues evident in the X-ray model of subtilisin Novo.

Energy Transfer↗
PubMed2005

Carbon-13 NMR method for the detection of correlated hydrogen exchange at adjacent backbone peptide amides and its application to hydrogen exchange in five antiparallel beta strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI).

A novel method for monitoring proton-deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the (13)C NMR signals from peptide carbonyls. The line shape of the carbonyl (13)C(i) signal is influenced by differential H/D occupancy at the two adjacent amides: the H(N)(i)(+1) (beta site) and the H(N)(i) (gamma site). At a carbon frequency of 75.4 MHz, the H --> D isotope shifts on the (13)C signal are about 5-7 Hz for exchange at the beta site and 2 Hz or less for exchange at the gamma site. Because the effects at the two sites are additive, the time dependence of the line shape of a particular carbonyl resonance can report not only the exchange rates at the individual sites but also the level of dual exchange. Therefore, the data can be analyzed to determine the rate (k(c)) and degree of correlated exchange (X(betagamma)) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues in Streptomyces subtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective (13)C' labeling at all valyl residues and one with selective (13)C' labeling at all leucyl residues. This permitted the direct observation by one-dimensional (13)C NMR of selected carbonyl signals from residues with slowly exchanging amides at the i and i + 1 positions. The residues investigated were located in an alpha helix and in a five-stranded antiparallel beta sheet. Samples of the two labeled proteins were prepared at various pH values, and (13)C NMR spectra were collected at 50 degrees C prior to and at various times after transferring the sample from H(2)O to (2)H(2)O. Most of the slowly exchanging amides studied were intramolecular hydrogen-bond donors. In agreement with prior studies, the results indicated that the exchange rates of the amide hydrogens in proteins are governed not only by hydrogen bonding but also by other factors. For example, the amide hydrogen of Thr34 exchanges rapidly even though it is an intramolecular hydrogen-bond donor. Over nearly the whole pH range studied, the apparent rates of uncorrelated exchange (k(beta) and k(gamma)) were proportional to [OH(-)] and the apparent rates of correlated exchange at two adjacent sites (k(c)) were roughly proportional to [OH(-)](2). This enabled us to extract the pH-independent exchange rates (k(beta) degrees , k(gamma) degrees , and k(c) degrees ). In all cases in which correlated exchange could be measured, the observed sigmoidal pH dependence of X(betagamma) could be replicated roughly from the derived pH-independent rates.

Bacterial Proteins↗
PubMed1996

Subtilisin BPN' variants: increased hydrolytic activity on surface-bound substrates via decreased surface activity.

Site-directed mutagenesis and random mutagenesis were used to produce variants of subtilisin BPN' (Bacillus amyloliquefaciens) protease with variable surface adsorption properties. Protease adsorption and peptide hydrolysis rate were measured for these variants using a model substrate consisting of a peptide covalently bound to a surface. While most variants adsorb at a level very similar to that of native BPN', several variants were identified which adsorb either more or less. For surface-bound substrates we report a linear dependence between the concentration of adsorbed protease enzyme and substrate hydrolysis, similar to the linear dependence between enzyme solution concentration and hydrolysis of soluble substrates. On the basis of this knowledge we hypothesized that variants designed to adsorb at a higher level on a surface-bound peptide substrate would hydrolyze that surface-bound substrate faster. Contrary to our original expectations, the variants that adsorb more on the covalently bound peptide surface hydrolyze this substrate slower. In addition, variants of BPN' which adsorb at a lower level than native BPN' hydrolyze the surface-bound substrate faster. Enzyme adsorption and the subsequent peptide hydrolysis are altered by substituting amino acids that modify the surface charge or hydrophobicity of the native enzyme. This effect is most dramatic when the changes were made at surface-exposed sites around the binding pocket/active site of the enzyme. One mechanism that is consistent with the data is based on the relationship between the level of adsorption and the enzyme's affinity for the surface. In this mechanism weakly adsorbed enzymes are postulated to move more rapidly from site to site on the surface, thereby increasing substrate hydrolysis.

Adsorption↗
PubMed1996

Increased proteolytic processing of protein tyrosine phosphatase mu in confluent vascular endothelial cells: the role of PC5, a member of the subtilisin family.

Cleavage and subsequent release of the extracellular domains of receptor protein tyrosine phosphatases (RPTP) occur at high cell density and may have an important role in regulating their activity. Because cleavage of RPTP occurs at a target motif (RXK/RR) recognized by a family of subtilisin/kexin-like endoproteases, we postulated that members of the subtilisin family may have an important role in this cleavage. We show in this report that the membrane-associated RPTPmu--both in its full 200-kDa form and as a 100-kDa cleavage product--is upregulated 4- and 7-fold, respectively, as human umbilical vein endothelial cells (HUVEC) approach confluence. To determine whether RPTPmu cleavage depended on PC5 (a subtilisin/kexin like endoprotease present in endothelial cells), we transfected COS cells with expression plasmids coding for RPTPmu and PC5 or the closely related protease PACE4. PC5, but not PACE4, cleaved RPTPmu, and RPTPmu cleavage was absent in COS cells transfected with an expression plasmid encoding a mutant PC5 whose active-site serine had been mutated to alanine. We also performed RNA blot analysis to determine whether PC5 expression was affected by confluence in HUVEC. PC5 mRNA levels were upregulated by more than 30-fold when confluence in HUVEC increased from 25% to 100%. These results indicate that PC5 may have an important role in mediating the cleavage of RPTPmu in response to contact inhibition in HUVEC.

Amino Acid Sequence↗
PubMed1998

Site-directed mutagenesis combined with chemical modification as a strategy for altering the specificity of the S1 and S1' pockets of subtilisin Bacillus lentus.

By combining site-directed mutagenesis with chemical modification, we have altered the S1 and S1' pocket specificity of subtilisin Bacillus lentus (SBL) through the incorporation of unnatural amino acid moieties, in the following manner: WT --> Cysmutant + H3CSO2SR --> Cys-SR, where R may be infinitely variable. A paradigm between extent of activity changes and surface exposure of the modified residue has emerged. Modification of M222C, a buried residue in the S1' pocket of SBL, caused dramatic changes in kcat/KM, of an up to 122-fold decrease, while modification of S166C, which is located at the bottom of the S1 pocket and is partially surface exposed, effected more modest activity changes. Introduction of a positive charge at S166C does not alter kcat/KM, whereas the introduction of a negative charge results in lowered activity, possibly due to electrostatic interference with oxyanion stabilization. Activity is virtually unaltered upon modification of S156C, which is located toward the bottom of the S1 pocket and surface exposed and whose side chain is solvated. An unexpected structure-activity relationship was revealed for S166C-SR enzymes in that the pattern of activity changes observed with increasing steric size of R was not monotonic. Molecular modeling analysis was used to analyze this unprecedented structure-activity relationship and revealed that the position of the beta-carbon of Cys166 modulates binding of the P1 residue of the AAPF product inhibitor.

Amino Acid Substitution↗
PubMed1998

The 0.78 A structure of a serine protease: Bacillus lentus subtilisin.

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.

Aspartic Acid↗
PubMed1999

Toward tailoring the specificity of the S1 pocket of subtilisin B. lentus: chemical modification of mutant enzymes as a strategy for removing specificity limitations.

In both protein chemistry studies and organic synthesis applications, it is desirable to have available a toolbox of inexpensive proteases with high selectivity and diverse substrate preferences. Toward this goal, we have generated a series of chemically modified mutant enzymes (CMMs) of subtilisin B. lentus (SBL) possessing expanded S(1) pocket specificity. Wild-type SBL shows a marked preference for substrates with large hydrophobic P(1) residues, such as the large Phe P(1) residue of the standard suc-AAPF-pNA substrate. To confer more universal P(1) specificity on S(1), a strategy of chemical modification in combination with site-directed mutagenesis was applied. For example, WT-SBL does not readily accept small uncharged P(1) residues such as the -CH(3) side chain of alanine. Accordingly, with a view to creating a S(1) pocket that would be of reduced volume providing a better fit for small P(1) side chains, a large cyclohexyl group was introduced by the CMM approach at position S166C with the aim of partially filling up the S(1) pocket. The S166C-S-CH(2)-c-C(6)H(11) CMM thus created showed a 2-fold improvement in k(cat)/K(M) with the suc-AAPA-pNA substrate and a 51-fold improvement in suc-AAPA-pNA/suc-AAPF-pNA selectivity relative to WT-SBL. Furthermore, WT-SBL does not readily accept positively or negatively charged P(1) residues. Therefore, to improve SBL's specificity toward positively and negatively charged P(1) residues, we applied the CMM methodology to introduce complementary negatively and positively charged groups, respectively, at position S166C in S(1). A series of mono-, di-, and trinegatively charged CMMs were generated and all showed improved k(cat)/K(M)s with the positively charged P(1) residue containing substrate, suc-AAPR-pNA. Furthermore, virtually arithmetic improvements in k(cat)/K(M) were exhibited with increasing number of negative charges on the S166C-R side chain. These increases culminated in a 9-fold improvement in k(cat)/K(M) for the suc-AAPR-pNA substrate and a 61-fold improvement in suc-AAPR-pNA/suc-AAPF-pNA selectivity compared to WT-SBL for the trinegatively charged S166C-S-CH(2)CH(2)C(COO(-))(3) CMM. Conversely, the positively charged S166C-S-CH(2)CH(2)NH(3)(+) CMM generated showed a 19-fold improvement in k(cat)/K(M) for the suc-AAPE-pNA substrate and a 54-fold improvement in suc-AAPE-pNA/suc-AAPF-pNA selectivity relative to WT-SBL.

Bacillus↗
PubMedDate not supplied

Supercritical fluids are superior media for catalysis by cross-linked enzyme microcrystals of subtilisin Carlsberg.

We report on the performance of cross-linked enzyme microcrystals (CLECs) of subtilisin Carlsberg in supercritical fluids (SC-fluids). The catalytic activity of CLECs in SC-ethane was found to be 2- to 10-fold greater than in hexane under the same conditions, using CLECs dried by propanol washing. Air-dried CLECs and lyophilized powders showed much lower activities, reflecting the same hydration hysteresis effects as in organic solvents. Reaction rates were much lower in SC-CO(2), especially at higher water activity, probably as a result of acid-base effects of carbonic acid on the enzyme.

Catalysis↗
PubMedDate not supplied

High initial activity but low storage stability observed for several preparations of subtilisin Carslberg suspended in organic solvents.

Colyophilization with methyl-beta-cyclodextrin activates subtilisin Carlsberg by more than 200-fold in organic solvents, though this is a short-lived effect. About 93% of the enzyme's high initial activity observed in THF (at 45 degrees C) decreases exponentially with a t(1/2) of 1.8 h, until it reaches a residual activity (of 7%) that remains constant throughout the 4 days duration of the experiment. A further study of this enzyme reveals a general trend: the initial activities of the lyophilized powder and the cross-linked enzyme crystals are also greatly reduced upon incubation in this solvent, although these preparations retain 50% of their activity after about 20 h of incubation. All of the preparations studied retained some residual activity (which persisted throughout the duration of the experiments) after the initial exponential decay. The data here presented suggest that the mode of enzyme preparation is an important issue to consider when planning lengthy reactions.

Enzyme Stability↗
PubMed2002

Coagulation of soy protein isolates induced by subtilisin Carlsberg.

The coagulation of soy protein isolates (SPI) induced by subtilisin Carlsberg was studied. The proteins were digested to fragments of 16 kDa or less in the early stage of the reaction, followed by coagulation. The time-course of the coagulation measured by turbidity was separated into three phases. The turbidity decreased from the initial level observed at time zero to the minimum level (OD1) at time T1 (15-20 min) in the first phase. Then, it increased drastically to reach the maximum (OD2) at time T2 (60-70 min) in the second phase, which was followed by a slight decrease in the third phase. The coagulation was terminated at T2, where 30-35% of the weight of the SPI proteins was in coagula. Proteins in the coagula were degraded slowly in the prolonged incubation, and the protein content in the coagula was finally 15-20% of the weight. The time-course of the turbidity agreed well with that of the weight of the precipitates formed, indicating that the turbidity reflects the progress of the coagulation. The turbidity change (OD1 to OD2) from the start to the end of the coagulation increased proportionally to the SPI concentration (4.9-11 mg/mL), although the time (T1 to T2) needed for the coagulation was independent of the concentration. The growth of the coagula is promoted by increasing the SPI concentration and is rate-limiting in the coagulation.

Electrophoresis, Polyacrylamide Gel↗
PubMed2004

Insights into the reaction mechanism of the coagulation of soy protein isolates induced by subtilisin carlsberg.

The reaction mechanism of the coagulation of soy protein isolates (SPIs) induced by subtilisin Carlsberg was investigated. Formation of the coagula was monitored by measuring the turbidity (OD660) of the SPI solution, which decreased at the initial stage (phase 1 or digestion phase) of the reaction, and then increased (phase 2 or coagulation phase) and finally reached the plateau level. The velocity of the coagulation increased with increasing enzyme concentration. The coagulation was inhibited dramatically by adding a serine protease inhibitor (phenylmethanesulfonyl fluoride, PMSF) when the turbidity reached the minimum value. This indicates that the SPI digests participating in the coagulation are produced mainly in phase 2; in other words, production of the coagulating fragments and their coagulation occur simultaneously in phase 2. Structural changes of SPI during proteolysis were measured by observing fluorescence changes of aromatic amino acids of SPI and an externally added hydrophobic probe. It was suggested that the hydrophilic surface areas of SPIs might be cleaved preferentially in phase 1, and that the hydrophobic inner areas might be cleaved in phase 2 with extensive decomposition of the 3-D structure of SPI proteins. The fragments formed in phase 2 are considered to coagulate through hydrophobic interactions.

Amino Acids, Aromatic↗
PubMed2001

Subtilisin-catalyzed synthesis of amino acid and peptide esters. Application in a two-step enzymatic ligation strategy.

We describe an efficient enzymatic approach to the synthesis of amino acid and peptide esters. The serine protease subtilisin Carlsberg (EC 3.4.21.62) was found to efficiently catalyze the specific formation of C(alpha)-carboxyl 3-hydroxypropyl or 4-hydroxybutyl esters of certain Boc-amino acids and peptides in high-content 1,3-propanediol or 1,4-butanediol solution, with substrate specificity parallel to that of the normal hydrolytic reaction. This approach can be coupled with kinetic-control reverse proteolysis in a two-step enzymatic peptide ligation scheme. [reaction: see text]

Amino Acids↗
PubMed2001

Backbone dynamics of the natively unfolded pro-peptide of subtilisin by heteronuclear NMR relaxation studies.

The dynamics of the natively unfolded form of the pro-peptide of subtilisin (PPS) have been characterized at two different pHs (6.0 and 3.0) by 15N relaxation experiments. 15N relaxation data is obtained at multiple field strengths and a detailed comparison of spectral density mapping, the model free approach and the recently proposed Cole-Cole model free (CC-MF) analysis is presented. The CC-MF analysis provides a better fit to the observed magnetic field dependence of 15N relaxation data of unfolded PPS than conventional model free approaches and shows that fluctuations in R2 may be accounted for by a distribution of correlation times on the nanosecond timescale. A new parameter epsilon derives from the analysis and represents the width of the distribution function and the heterogeneity of the dynamics on the nanosecond timescale at a particular site. Particularly interesting is the observation that epsilon is sensitive to pH changes and that PPS samples a wider distribution of nanosecond time scale motions at less acidic pHs than at more acidic pHs. These results suggest that PPS experiences a higher degree of correlated motion at pH 6.0 and that electrostatic interactions may be important for inducing correlated motions on the nanosecond timescale in unfolded PPS.

Amino Acid Sequence↗
PubMed1997

Interaction of phomopsin A with normal and subtilisin-treated bovine brain tubulin.

Tubulin, the major component of microtubules, has a tendency to lose its ability to assemble or to bind to ligands in a time-dependent process known as "decay." The decay process also causes tubulin to expose sulfhydryl groups and hydrophobic areas. The antimitotic drug phomopsin A strongly protects the tubulin molecule from decay. Here we have studied the interaction of phomopsin A with alpha beta tubulin and tubulin which has been treated with subtilisin to remove selectively the C-termini of the alpha and beta chains (alpha(s) beta(s)). The binding of phomopsin A to alpha beta tubulin decreases the sulfhydryl titer by approximately 1.0 mol/mol. Selective removal of the peptides from the C-terminal ends does not affect phomopsin A's interaction with tubulin. Moreover, the alpha(s) beta(s) tubulin-phomopsin A complex appears to be more stable than the alpha bet tubulin-phomopsin A complex as determined by the time-dependent increase in exposure of sulfhydryl groups and hydrophobic areas on tubulin. In fact, phomopsin A inhibits the decay process of alpha(s) beta(s) tubulin completely. This observation raises the possibility of determining the conformation of this configuration of tubulin.

Alkylation↗
PubMed2004

Subtilisin protein inhibitor from potato tubers.

A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 +/- 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.

Amino Acid Sequence↗
PubMed2001

Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes.

The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions. Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0-1.4 A resolution. The structure of PSCP comprises a single compact domain with a diameter of approximately 55 A, consisting of a seven-stranded parallel beta-sheet flanked on both sides by a number of helices. The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue. Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287.

Amino Acid Sequence↗
PubMed1993

Processing of protein precursors by a novel family of subtilisin-related mammalian endoproteases.

The recent identification of a novel family of mammalian endoproteases that carry out intracellular processing of protein precursors at dibasic sites has ended a search that began twenty-five years ago with the discovery of the first such precursor, proinsulin. The five proteases found thus far are all related to the yeast dibasic-specific endoprotease kex2, and include PC2, PC3/PC1, PC4, furin/PACE, and PACE4. All are Ca(2+)-dependent serine proteases with catalytic domains organized similarly to the bacterial subtilisins. The emerging characteristics of these endoproteases, including their tissue-specific expression, subcellular localization, and cleavage site selectivity, indicates that members of this family arose during evolution to process a diverse group of functionally distinct precursors in a highly specific, compartmentalized and regulated fashion.

Amino Acid Sequence↗
PubMed2000

Direct on-filter assay of subtilisin-type proteolytic enzymes for rapid analysis of analyte captured from the workplace atmosphere.

A simple assay for some proteolytic enzymes has been developed which can be performed directly on the surface of a cellulose nitrate filter used to capture the analyte during workplace monitoring for health and safety purposes. Following air sampling the analysis is performed on the filter which is retained within the air sampler. This involves two steps: first, a 15 min incubation in which the captured enzyme is dissolved and then digests an alkaline-phosphatase-labelled antibody immobilised as a small dot on the surface of the filter; and second, is a 10 min incubation with substrate solution, which follows an in situ wash under a vacuum. During the incubation colour develops on the spot at the location of the immobilised enzyme antibody conjugate. The intensity of the spot can be assessed visually within the sampler to ascertain the presence or absence of captured enzyme, or alternatively quantitative results can be obtained using an optical scanner. The limit of detection is 5 ng per filter for subtilisin (20 ng for visual discrimination between this standard and the zero). The assay is stable to the effects of ambient air sampling at 31 min(-1) for 18 h.

Air Pollution, Indoor↗