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PubMed1992

Energetics of folding subtilisin BPN'.

Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. Here we document for the first time the in vitro folding of the mature form of subtilisin. Subtilisin was modified by site-directed mutagenesis to be proteolytically inactive, allowing the impediments to folding to be systematically examined. First, the thermodynamics and kinetics of calcium binding to the high-affinity calcium A-site have been measured by microcalorimetry and fluorescence spectroscopy. Binding is an enthalpically driven process with an association constant (Ka) equal to 7 x 10(6) M-1. Furthermore, the kinetic barrier to calcium removal from the A-site (23 kcal/mol) is substantially larger than the standard free energy of binding (9.3 kcal/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow (t1/2 = 1.3 h at 25 degrees C). Second, to measure the kinetics of subtilisin folding independent of calcium binding, the high-affinity calcium binding site was deleted from the protein. At low ionic strength (I = 0.01) refolding of this mutant requires several days. The folding rate is accelerated almost 100-fold by a 10-fold increase in ionic strength, indicating that part of the free energy of activation may be electrostatic. At relatively high ionic strength (I = 0.5) refolding of the mutant subtilisin is complete in less than 1 h at 25 degrees C. We suggest that part of the electrostatic contribution to the activation free energy for folding subtilisin is related to the highly charged region of the protein comprising the weak ion binding site (site B).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1991

Probing the mechanism and improving the rate of substrate-assisted catalysis in subtilisin BPN'.

A mutant of the serine protease, subtilisin BPN', in which the catalytic His64 is replaced by Ala (H64A), is very specific for substrates containing a histidine, presumably by the substrate-bound histidine assisting in catalysis [Carter, P., & Wells, J.A. (1987) Science (Washington, D.C.) 237, 394-399]. Here we probe the catalytic mechanism of H64A subtilisin for cleaving His and non-His substrates. We show that the ratio of aminolysis to hydrolysis is the same for ester and amide substrates as catalyzed by the H64A subtilisin. This is consistent with formation of a common acyl-enzyme intermediate for H64A subtilisin, analogous to the mechanism of the wild-type enzyme. However, the catalytic efficiencies (kcat/KM) for amidase and esterase activities with His-containing substrates are reduced by 5000-fold and 14-fold, respectively, relative to wild-type subtilisin BPN, suggesting that acylation is more compromised than deacylation in the H64A mutant. High concentrations of imidazole are much less effective than His substrates in promoting hydrolysis by the H64A variant, suggesting that the His residue on the bound (not free) substrate is involved in catalysis. The reduction in catalytic efficiency kcat/KM for hydrolysis of the amide substrate upon replacement of the oxyanion stabilizing asparagine (N155G) is only 7-fold greater for wild-type than H64A subtilisin. In contrast, the reductions in kcat/KM upon replacement of the catalytic serine (S221A) or aspartate (D32A) are about 3000-fold greater for wild-type than H64A subtilisin, suggesting that the functional interactions between the Asp32 and Ser221 with the substrate histidine are more compromised in substrate-assisted catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
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↗
PubMed2001

Stabilizing mutations and calcium-dependent stability of subtilisin.

Stability is a property of subtilisin which has proven particularly amenable to enhancement via random mutagenesis and screening, yet the effects of most stabilizing mutations are not understood in structural and energetic detail. This paper seeks to explain the longstanding observation that stabilizing mutations are usually calcium-dependent in their stabilizing effect, irrespective of their proximity to the calcium binding sites. Stabilizing mutations in subtilisin fall into one of three classes. The largest class of mutations stabilize only in the presence of excess calcium. A smaller number of mutations stabilize independently of [calcium], and a few mutations stabilize only in the presence of chelating agents, such as EDTA. This study compares the effects of mutations from each class when introduced into subtilisin BPN' and two calcium-free versions of subtilisin. The calcium-dependent effects of mutations can be explained by considering subtilisin to be in conformational equilibrium between two structurally similar but energetically distinct states: N and N*. The equilibrium from the N* to the N state can be altered either by calcium binding to site A or by mutation. Mutations which stabilize only in the presence of calcium stabilize the N state relative to N*. Mutations which stabilize only in the presence of chelants stabilize the N* state relative to N. As a byproduct of this analysis, we have developed a hyperstable variant of subtilisin whose inactivation at high temperature in the presence of EDTA is 10(5) times slower than wild-type subtilisin.

Bacillus↗
PubMed1996

Furilisin: a variant of subtilisin BPN' engineered for cleaving tribasic substrates.

The serine protease, subtilisin BPN', was engineered to cleave proteins after tribasic sequences in a manner that resembles the substrate specificity of furin, one of the mammalian subtilisin homologs that processes prohormones. As a starting point we used a double mutant of subtilisin BPN' (N62D/ G166D) that showed substantial preference for cleaving after sequences having consecutive dibasic residues (namely, at the P1 and P2 substrate positions) [Ballinger et al. (1995) Biochemistry 34, 13312-13319]. Additional specificity for basic residues was engineered at the P4 position by introducing subtilisin-to-furin substitutions at three hydrophobic residues that composed the S4 subsite (Y104, I107, and L126). Initial attempts to incorporate a Y104D or I107E mutation or the Y104D/I107E double mutation into the dibasic specific enzyme failed to generate the processed enzyme. The problem was traced to the inability of the mutant prosubtilisins to process themselves and fold correctly. Replacing the natural processing site sequence (AHAY) with a good furin substrate sequence (RHKR) resulted in expression of the triple subtilisin mutant (N62D/Y104D/G166D) we call "furilisin". Furilisin hydrolyzes synthetic tribasic substrates (succinyl-RAKR-pNA or succinyl-KAKR-pNA) with high catalytic efficiency (kcat/K(m) > 3 x 10(5) M-1 s-1) and discriminates in favor of Arg versus Ala at the P4 position by a factor of 360. The overall specificity change versus the wild-type enzyme was dramatic. For example, succinyl-RAKR-pNA was cleaved approximately 60000 times faster than succinyl-AAPF-pNA, a good substrate for wild-type subtilisin. Similarly, furilisin was inhibited (K1* = 29 nM) by a variant of the turkey ovomucoid third domain inhibitor that contained an engineered furin substrate site (RCKR decreases) [Lu et al. (1993) J. Biol. Chem. 268, 14583-14585] and not by one having a good wild-type subtilisin substrate sequence (ACTL decreases). Interestingly, the extreme changes in substrate specificity resulted from substantial synergy between the engineered subsites. These studies provide a basic example of how to manipulate substrate specificity in a modular fashion, thereby creating an engineered-enzyme that may be useful as a protein processing tool.

Amino Acid Sequence↗
PubMed1996

A low-barrier hydrogen bond in subtilisin: 1H and 15N NMR studies with peptidyl trifluoromethyl ketones.

The N delta 1 proton of His 64 forms a hydrogen bond with Asp 32, as part of the catalytic triad in serine proteases of the subtilisin family. His 64 in subtilisin has been studied by 1H and 15N NMR spectroscopy in the presence and absence of peptidyl trifluoromethyl ketones (TFMKs) that are transition state analog inhibitors. For subtilisin Carlsberg, the downfield resonance of the imidazolium N delta 1 proton is approximately 18.3 ppm and the D/H fractionation factor is 0.55 +/- 0.04 at pH 5.5 (11 degrees C), and 0.63 +/- 0.04 (5 degrees C) and 0.68 +/- 0.04 at pH 6 (11 degrees C). In the complex between subtilisin Carlsberg and Z-L-leucyl-L-leucyl-L-phenylalanyltrifluoromethyl ketone (Z-LLF-CF3) at pH values between 6.5 and 10.6, His 64 remains positively charged, and the D/H fractionation factor of its N delta 1 proton is 0.85 +/- 0.05. In the complex between a subtilisin variant from Bacillus lentus and Z-LLF-CF3, the proton resonance at 18.8 ppm is correlated with a 15N resonance at 197.6 ppm downfield from liquid NH3 with a 1JNH of 81 Hz. The chemical shifts of subtilisin complexes with peptidyl TFMKs are among the most downfield shifts reported for any protein. At pH 9.5, His 64 is neutral and the D/H fractionation factor increases to 1.2 with a chemical shift of 15.0. His 64 is positively charged in the free enzyme at low pH, the inhibitor hemiketal complex at neutral pH, and the transition state for amide bond hydrolysis. These data thus provide indirect evidence for the presence of a low-barrier hydrogen bond in the catalytic mechanism of subtilisin proteases.

Bacterial Proteins↗
PubMed2004

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal beta-barrel domain.

The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering. The native form was refined to a crystallographic R factor of 0.134 (Rfree of 0.169) at 1.30-A resolution. KP-43 consists of two domains, a subtilisin-like alpha/beta domain and a C-terminal jelly roll beta-barrel domain. The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal beta-barrel domain both differ in each case. Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role. KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases. To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (Rfree of 0.212) at 1.73-A resolution. The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN'. Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70-80% activity against proteinaceous substrates. These results, as well as the beta-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.

Animals↗
PubMed1989

The role of Pro-239 in the catalysis and heat stability of subtilisin E.

Site-directed mutagenesis was employed to analyze the role of an alpha-helix containing catalytic Ser-221 of subtilisin E. Pro-239 located at the carboxy-terminal end of the alpha-helix was first replaced with Gly to examine the role of Pro-239 in the catalysis and stability of subtilisin E. The mutation was found to decrease both the catalytic rate (kcat) and the heat stability. This result strongly suggests that Pro-239 plays an important role in the maintenance of the alpha-helix, affecting the functioning of the active site. Various amino acid substitutions at position 239 were attempted to obtain the active subtilisins from Gly-239 subtilisin. Lys- and Arg-substitutions were found to result in more active and stable subtilisins than the Gly-239 subtilisin. In particular, the Arg-239 mutant showed enhanced heat stability compared with the wild type. These results demonstrate the important role of the alpha-helix containing catalytic Ser-221 in the catalysis as well as in the heat stability of subtilisin.

Catalysis↗
PubMed1995

The 2 A crystal structure of subtilisin E with PMSF inhibitor.

Using enzyme prepared by the DNA recombination technique, subtilisin E from Bacillus subtilis was crystallized in space group P2(1)2(1)2(1) with two molecules in an asymmetric unit. The crystal structure of PMSF-inhibited subtilisin E was solved by molecular replacement followed by refinement with the X-PLOR program. This resulted in the 2.0 A structure of subtilisin E with an R-factor of 0.191 for 8-2 A data and r.m.s. deviations from ideal values of 0.021 A and 2.294 for bond lengths and bond angles respectively. The PMSF group covalently bound to Ser221 appeared very clearly in the electron density map. Except for the active site disturbed by PMSF binding, the structural features of subtilisin E are almost the same as in other subtilisins. The calcium-binding sites are different in detail in the two independent molecules of subtilisin E. Based on the structure, the remarkably enhanced heat stability of mutant N118S of subtilisin E is discussed. It is very likely that there is an additional water molecule in the mutant structure, which is hydrogen bonded to side chains of Ser118 and its neighbouring residues Lys27 and Asp120.

Amino Acid Sequence↗
PubMed1990

A highly active and oxidation-resistant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chemical modification.

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1-10% of that obtained with the wild-type enzyme. In contrast, the oxidation-labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222. It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site-directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild-type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me-S-Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild-type enzyme when assayed against the substrate Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc-Ala-Ala-Pro-Phe-NH-Ph-NO2. This demonstrates the feasibility of replacing the oxidation-labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.

Bacillus↗
PubMed2000

Subtilisin increases macromolecular efflux from the oral mucosa.

The purpose of this study was to determine whether subtilisin, a potent serine proteinase derived from Bacillus species contaminating smokeless tobacco, increases macromolecular efflux from the oral mucosa and, if so, whether local elaboration of bradykinin mediates this response. Using intravital microscopy, I found that suffusion of subtilisin elicits significant, concentration-dependent leaky site formation and an increase in the clearance of fluorescein isothiocyanate-labeled dextran (molecular mass, 70 kDa) from the in situ hamster cheek pouch (P<0.05). Heat-inactivated subtilisin had no significant effects on macromolecular efflux. Subtilisin-induced responses were significantly attenuated by Hoe 140 and NPC 17647, two structurally distinct selective bradykinin B(2) receptor antagonists, but not by des-Arg(9)-[Leu(8)]bradykinin, a selective bradykinin B(1) receptor antagonist, or CP-96,345, a selective neurokinin-1 receptor antagonist. Aprotinin, but not leupeptin, significantly attenuated subtilisin-induced increase in macromolecular efflux. Indomethacin had no significant effects on subtilisin-induced responses. Collectively, these data indicate that subtilisin increases the macromolecular efflux from the in situ hamster cheek pouch in a catalytic-site-dependent fashion through local elaboration of bradykinin. This response does not involve the stimulation of local afferent nerves or the production of prostaglandins.

Animals↗
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↗
PubMed1988

Engineering thermostability in subtilisin BPN' by in vitro mutagenesis.

A procedure has been developed for the isolation and identification of mutants of the bacterial serine protease, subtilisin, which exhibit enhanced thermostability. The cloned subtilisin BPN' gene from Bacillus amyloliquefaciens was treated with a variety of chemical mutagens to introduce random mutations in the coding sequence. Strains containing the cloned, mutagenized subtilisin gene which produced subtilisin with enhanced thermostability were selected by a simple plate assay procedure, which screens for esterase activity on nitrocellulose filters after preincubation at elevated temperatures. The identification and characterization of eight different stabilizing mutations are described. Several mutants containing various combinations of these stabilizing mutations were constructed by oligonucleotide-directed mutagenesis. Combining independent, stabilizing mutations in the same subtilisin molecule has resulted in an approximate multiplicative decrease in the rate of thermal inactivation. In this way, a variant of subtilisin has been constructed which is about 12-fold more stable than wild-type subtilisin, with no radical changes in the tertiary protein structure but rather minor, independent alterations in amino acid sequence. The ultimate goal in these studies is to be able to accurately predict where stabilizing changes can be made in a protein.

Amino Acid Sequence↗
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↗
PubMed1994

Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41.

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility.

Amino Acid Sequence↗
PubMed1998

Crystal structure of calcium-independent subtilisin BPN' with restored thermal stability folded without the prodomain.

The three-dimensional structure of a subtilisin BPN' construct that was produced and folded without its prodomain shows the tertiary structure is nearly identical to the wild-type enzyme and not a folding intermediate. The subtilisin BPN' variant, Sbt70, was cloned and expressed in Escherichia coli without the prodomain, the 77-residue N-terminal domain that catalyzes the folding of the enzyme into its native tertiary structure. Sbt70 has the high-affinity calcium-binding loop, residues 75 to 83, deleted. Such calcium-independent forms of subtilisin BPN' refold independently while retaining high levels of activity [Bryan et al., Biochemistry, 31:4937-4945, 1992]. Sbt70 has, in addition, seven stabilizing mutations, K43N, M50F, A73L, Q206V, Y217K, N218S, Q271E, and the active site serine has been replaced with alanine to prevent autolysis. The purified Sbt70 folded spontaneously without the prodomain and crystallized at room temperature. Crystals of Sbt70 belong to space group P2(1)2(1)2(1) with unit cell parameters a = 53.5 A, b = 60.3 A, and c = 83.4 A. Comparison of the refined structure with other high-resolution structures of subtilisin BPN' establishes that the conformation of Sbt70 is essentially the same as that previously determined for other calcium-independent forms and that of other wild-type subtilisin BPN' structures, all folded in the presence of the prodomain. These findings confirm the results of previous solution studies that showed subtilisin BPN' can be refolded into a native conformation without the presence of the prodomain [Bryan et al., Biochemistry 31:4937-4945, 1992]. The structure analysis also provides the first descriptions of four stabilizing mutations, K43N, A73L, Q206V, and Q271E, and provides details of the interaction between the enzyme and the Ala-Leu-Ala-Leu tetrapeptide found in the active-site cleft.

Binding Sites↗
PubMed1993

Multiple sites for subtilisin cleavage of tubulin: effects of divalent cations.

Limited digestion of pig brain GDP-tubulin by subtilisin was carried out in the presence of Mg2+, Mn2+, Ca2+, Zn2+, or Be2+. Isoelectric focusing, followed by SDS-PAGE, revealed characteristic divalent cation-dependent changes in the alpha- and beta-tubulin cleavage patterns. Previous studies revealed that the beta-cleavage pattern is different for heterodimers and microtubules [Lobert and Correia, 1992: Arch. Biochem. Biophys. 296: 152-160]. Divalent cation effects on subtilisin digestion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and electron microscopy reveal that GDP-tubulin cleaved by subtilisin in the presence of Mg2+, Ca2+, or Mn2+ forms sheets of rings. Mn2+ induces ring formation in uncleaved GDP-tubulin. Isotype-depleted tubulin was generated by the removal of class III beta-tubulin using immunoaffinity chromatography. Subtilisin digestion of the depleted fraction and the purified class III beta-tubulin demonstrates that cleavage occurs at three to four distinct sites. Thus, subtilisin-digested tubulin is more heterogeneous than was previously reported and the cleavage sites depend on solution conditions, divalent cations, and the state of assembly. This has important implications for experiments that utilize subtilisin-digested tubulin for studying microtubule-associated protein binding.

Animals↗
PubMed1996

Interaction of subtilisins with serpins.

Serpins are well-characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that alpha 1 proteinase inhibitor inhibits subtilisin Carlsberg and proteinase K, and alpha 1 antichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0-45 degrees C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.

Amino Acid Sequence↗