Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Thermolysin”

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 127 records · Page 7Linked to original sources
PubMed1996

Analysis of structural determinants of the stability of thermolysin-like proteases by molecular modelling and site-directed mutagenesis.

The thermolysin-like protease (TLP) produced by Bacillus stearothermophilus CU21 (TLP-ste) differs at 43 positions from the more thermally stable thermolysin (containing 316 residues in total). Of these differences, 26 were analysed by studying the effect of replacing residues in TLP-ste by the corresponding residues in thermolysin. Several stabilizing mutations were identified but, remarkably, considerable destabilizing mutational effects were also found. A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertional difference between the two enzymes) appeared to make an important contribution to the stability of the enzyme. Mutations with large effects on stability were all localized in the beta-pleated N-terminal domain of TLP-ste, confirming observations that this domain has a lower intrinsic stability than the largely alpha-helical C-terminal domain. Rigidifying mutations such as Gly58-->Ala and Ala69-->Pro were among the most stabilizing ones. Apart from this observation, the analyses did not reveal general rules for stabilizing proteins. Instead, the results highlight the importance of context in evaluating the stability effects of mutations.

Amino Acid Sequence↗
PubMed1996

Differentiation of thermolysins and serralysins by monoclonal antibodies.

Two monoclonal antibodies (MAbs) to a 36-kDa extracellular metalloprotease (PSCP) from Burkholderia (Pseudomonas) cepacia were found to react with thermolysin, Pseudomonas aeruginosa elastase, alkaline protease (Apr) and LasA, Serratia marcescens protease (SMP), Aeromonas hydrophila protease (AhP), and both the lethal factor (LF) and protective antigen (PA) of Bacillus anthracis on immunoblots. The MAbs were capable of neutralising the proteolytic activity of thermolysin, P. aeruginosa elastase and PSCP but not that of Apr, SMP, and AhP. These results suggest that these MAbs may be able to differentiate between the thermolysin and serralysin family of metalloproteases on the basis of their neutralisation capability and could, therefore, be useful tools in the characterisation of new bacterial proteases.

Aeromonas hydrophila↗
PubMed2005

Preliminary X-ray crystallographic analysis of thermolysin in the presence of 4 M NaCl.

The activity of thermolysin (EC 3.4.24.27) is greatly enhanced by high concentrations of neutral salts. For instance, 4 M NaCl enhances the activity 13-15-fold [Holmquist & Vallee (1976), Biochemistry, 15, 101-107; Inouye (1992), J. Biochem. (Tokyo), 112, 335-340]. To clarify the structural basis of the activation of thermolysin by high concentrations of NaCl, we have developed a new method to introduce 4 M NaCl into the P6(1)22 crystal of thermolysin originally grown without NaCl. The crystal obtained by this method diffracted X-rays to 2.43 A. No unit-cell parameter change was observed except the length of the c axis, which was elongated by 9.6% by the introduction of 4 M NaCl.

Bacillus↗
PubMed1990

Semisynthesis of carboxy-terminal fragments of thermolysin.

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205-316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205-302 and 303-316. Upon incubation for 2-5 days of fragment 205-302 with a 5-fold excess of peptide 303-316, prepared by solid phase synthesis, with V8-protease in 0.1 M ammonium acetate, pH 6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to approximately 90% (based on fragment 205-302). The same procedure was used to prepare also the thermolysin fragments 205-315 and 205-311 by enzymatic coupling of fragment 205-302 to peptide 303-315 or 303-311, these last prepared by proteolytic digestion of the synthetic peptide 303-316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205-316 of thermolysin at the level of its helical segment encompassing residues 301-312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.

Amino Acid Sequence↗
PubMed1984

Pressure dependence of thermolysin catalysis.

A comparison of the pressure and temperature dependences of the catalytic reaction of thermolysin, a thermostable neutral protease from Bacillus thermoproteolyticus, with those of a non-thermostable neutral protease from Bacillus subtilis revealed a distinct difference in Km values of these enzymes for 3-(2-furyl)acrylyl-blocked dipeptide and tripeptide substrates, but not for Kcat. Namely, the volume changes for the binding process (delta V) for these substrates and several competitive inhibitors were -20- -30 ml/mol for thermolysin and nearly 0 ml/mol for the non-thermostable neutral protease. The enthalpy and entropy changes for the binding process were negative for thermolysin, but positive for the latter enzyme. The activation volumes (delta V not equal to) for the kcat process were 25 -35 ml/mol for both proteases, and activation enthalpy and entropy showed no significant difference between the two enzymes. The characteristic difference in the pressure and the temperature dependences seen for the binding process is discussed in relation to the thermostability of the proteases.

Bacillus subtilis↗
PubMed1986

Crystallographic structural analysis of phosphoramidates as inhibitors and transition-state analogs of thermolysin.

The mode of binding to thermolysin of the unsubstituted phosphoramidate inhibitor N-phosphoryl-L-leucinamide (P-Leu-NH2) has been determined crystallographically and refined at high resolution (R = 17.9% to 0.16-nm resolution). The mode of binding of the naturally occurring thermolysin inhibitor phosphoramidon reported previously [Weaver, L. H., Kester, W. R. and Matthews, B. W. (1977) J. Mol. Biol. 114, 119-132] has also been confirmed by crystallographic refinement (R = 17.4% to 0.23-nm resolution). Phosphoramidon binds to the enzyme with a single oxygen of the phosphoramidate moiety as a zinc ligand. Together with three ligands to the metal from the protein the resultant complex has approximately tetrahedral geometry. However, in the case of P-Leu-NH2, two of the phosphoramidate oxygens interact with the zinc to form a complex that tends towards pentacoordinate. In this respect, P-Leu-NH2 appears to be a better transition-state analog than is phosphoramidon. In addition, the phosphorus-nitrogen bond length in P-Leu-NH2 is 0.18 nm, suggesting that the nitrogen is protonated whereas the same bond in phosphoramidon is much shorter (0.15 nm) suggesting that the nitrogen does not carry a charge. In phosphoramidon the distance from the phosphoramide nitrogen to Glu-143 is 0.39 nm whereas in P-Leu-NH2 this distance decreases to 0.34 nm. Taken together, these observations provide additional evidence in support of the participation of pentacoordinate intermediates in the mechanism of action of thermolysin [Holmes, M. A. and Matthews, B. W. (1981) Biochemistry 20, 6912-6920] and the role of Glu-143 in first promoting the attack of a water molecule on the carbonyl carbon of the scissile bond and subsequently acting as a 'proton shuttle' to transfer the proton to the leaving nitrogen [Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5724-5729; Hangauer, D. G., Monzingo, A. F. and Matthews, B. W. (1984) Biochemistry 23, 5730-5741].

Crystallography↗
PubMed1993

Synergistic binding of hydrophobic probes and zinc ligands to thermolysin.

The strong synergism previously observed in the binding of inhibitors to two Zn-proteases, has also been found for thermolysin. As in earlier cases, the effects are produced by a small Zn-ligand (e.g. a hydroxamate) in the presence of another compound which contains the key structural features of specific substrates (a specificity probe). For thermolysin, the most effective specificity probes are hydrophobic derivatives of amines and amino acids (e.g. carbobenzyloxy-L-alaninol). Even the simple combination of benzyl alcohol and formohydroxamate displays considerable synergism. The above effects are temperature dependent and correlate well with a thermally induced conformational isomerization reported recently for this enzyme. Our results seem to be related to previous observations of substrate synergism in the reverse reaction and to superactivation by chemical modification of this enzyme. All these effects are consistent with a change in the environment of the catalytically important zinc atom upon binding of the hydrophobic side chain of the substrate. With the inclusion of thermolysin, binding synergism is now known to occur in an endopeptidase as well as in exopeptidases of diverse specificity. The general occurrence of this phenomenon in zinc proteases and its possible significance are discussed in an accompanying study.

Amines↗
PubMed1994

Thermolysin activation mutants with changes in the fusogenic region of an influenza virus hemagglutinin.

Influenza virus A/seal/Mass/1/80 (H7N7) mutants were obtained; the hemagglutinins (HAs) of the mutants were not activated by trypsin, as in the wild-type virus, but by thermolysin. The mutants grew efficiently under multiple replication cycle conditions and formed plaques in chicken embryo cells only when thermolysin was added to the culture medium. They exhibited hemolytic activity and induced protective immunity in chickens after an asymptomatic course of infection. Nucleotide sequencing of the HA gene and direct amino acid sequencing showed that insertion of a single leucine into the fusion peptide of the HA2 chain close to the cleavage site and a shift of the cleavage site toward the C terminus by one amino acid were responsible for the changes in the biological properties of the thermolysin activation mutants. Revertants could be obtained when trypsin or trypsin-like endoproteases were present in the virus-producing system.

Amino Acid Sequence↗
PubMed2002

Human bronchial epithelial cells express PAR-2 with different sensitivity to thermolysin.

Protease-activated receptor-2 (PAR-2) plays a role in inflammatory reactions in airway physiology. Proteases cleaving the extracellular NH(2) terminus of receptors activate or inactivate PAR, thus possessing a therapeutic potential. Using RT-PCR and immunocytochemistry, we show PAR-2 in human airway epithelial cell lines human bronchial epithelial (HBE) and A549. Functional expression of PAR-2 was confirmed by Ca(2+) imaging studies using the receptor agonist protease trypsin. The effect was abolished by soybean trypsin inhibitor and mimicked by the specific PAR-2 peptide agonist SLIGKV. Amplitude and duration of PAR-2-elicited Ca(2+) response in HBE and A549 cells depend on concentration and time of agonist superfusion. The response is partially pertussis toxin (PTX) insensitive, abolished by the phospholipase C inhibitor U-73122, and diminished by the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Cathepsin G altered neither the resting Ca(2+) level nor PAR-2-elicited Ca(2+) response. Thermolysin, a prototypic bacterial metalloprotease, induced a dose-dependent Ca(2+) response in HBE, but not A549, cells. In both cell lines, thermolysin abolished the response to a subsequent trypsin challenge but not to SLIGKV. Thus different epithelial cell types express different PAR-2 with identical responses to physiological stimuli (trypsin, SLIGKV) but different sensitivity to modifying proteases, such as thermolysin.

Bronchi↗
PubMed1998

Need for aromatic residue at position 115 for proteolytic activity found by site-directed mutagenesis of tryptophan 115 in thermolysin.

In thermolysin, tryptophan 115 seems to be at the S2 subsite. Trp-115 was replaced with tyrosine, phenylalanine, leucine, and valine during site-directed mutagenesis in order to evaluate the role of Trp-115 in the proteolytic activity of thermolysin. The mutant enzymes with Tyr-115 or Phe-115 had as much proteolytic activity as the wild-type enzyme, but the other two mutant enzymes had no activity. We found earlier that the substitution of Trp-115 with alanine, glutamic acid, lysine, and glutamine causes the enzyme to lose all activity, so an aromatic amino acid at position 115 seems to be essential for thermolysin.

Escherichia coli↗
PubMed1994

Redetermination and refinement of the complex of benzylsuccinic acid with thermolysin and its relation to the complex with carboxypeptidase A.

The complex of benzylsuccinic acid with thermolysin has been redetermined at 1.7-A resolution and refined to a crystallographic residual of 15.7%. In contrast to the prior study, which was to 2.3-A resolution, and without the benefit of refinement (Bolognesi, M. C. and Matthews, B. W. (1979) J. Biol. Chem. 254, 634-639), the present analysis shows that it is the D- rather than the L-isomer of benzylsuccinic acid that binds. The stereochemistry of the zinc-carboxylate interaction is now seen to be syn, as is also observed in all known zinc-carboxylate complexes of both thermolysin and carboxypeptidase A. The mode of binding of the beta-carboxylate resembles the presumed geometry of the tetrahedral transition state and, as such, is consistent with the commonly accepted mechanism of action of thermolysin and of carboxypeptidase A.

Binding Sites↗
PubMed1988

The binding of L-valyl-L-tryptophan to crystalline thermolysin illustrates the mode of interaction of a product of peptide hydrolysis.

Crystallographic analysis of the binding of mercaptoacetyl-L-valyl-L-tryptophan to thermolysin suggests that this inhibitor is hydrolyzed by the crystalline enzyme. The apparent product of hydrolysis, L-valyl-L-tryptophan (Val-Trp), occupies the S1'-S2' subsites of the active site, not the S1-S1' subsites as observed previously for the dipeptide L-alanyl-L-phenylalanine (Ala-Phe). The difference in binding of Val-Trp and Ala-Phe is consistent with the specificity requirements and preferences of thermolysin. The binding of Val-Trp illustrates the mode of interaction of one of the products of peptide hydrolysis. High resolution crystallographic refinement indicates that the valyl amino group makes three hydrogen bonds to the enzyme and to solvent and, in addition, is 2.8 A from the carboxylate of Glu-143. This is the first instance in which a direct interaction has been observed between Glu-143 and the scissile nitrogen. As such, the study directly supports the mechanism of action for thermolysin proposed by Hangauer et al. (Hangauer, D. G., Monzingo, A. F., and Matthews, B. W. (1984) Biochemistry 23, 5730-5741) and, by analogy, indirectly supports the similar mechanism proposed for carboxypeptidase A (Monzingo, A. F., and Matthews, B. W. (1984) Biochemistry 23, 5724-5729).

Binding Sites↗
PubMed1975

Use of thermolysin for the dissociation of lung tissue into cellular components.

A method is described for the dissociation of rat lungs into individual viable cells. Thermolysin was perfused through the vasculature and trachea. The lung was then minced and further dissociated by washing with sequential additions of thermolysin. The results indicate this procedure to be an effective means of dispersing lung tissue into its cellular components. Standard trypsinizing procedures generally yield approximately 5 per cent of the total available lung cells whereas the thermolysin treatment increases cell yield 10-fold without significantly affecting cell viability.

Animals↗
PubMed1992

Structural basis for the action of thermolysin.

High resolution X-ray crystallography has been used to determine the modes of binding to thermolysin of a series of different inhibitors including dipeptides, mercaptans, hydroxamates, N-carboxymethyl peptides and phosphonamidates. The interactions displayed by such inhibitors illustrate interactions that are presumed to occur between the enzyme and its substrates during catalysis. The crystallographic analysis, together with model building, suggest a detailed stereochemical mechanism of action for thermolysin and, by analogy, other zinc proteases such as carboxypeptidase A and the angiotensin converting enzyme. Analysis of a series of phosphonamidates, which are presumed to be transition-state analogues, has shown that chemically similar inhibitors can adopt dissimilar modes of binding. These different configurations provide a rationalization for large differences in the kinetics of binding that are observed for these inhibitors. Experiments with thermolysin as a test case suggest that knowledge of the three-dimensional structure of an enzyme or receptor will greatly facilitate the rational design of drugs directed at such targets.

Amino Acid Sequence↗
PubMed1986

Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin.

A comparison has been made of the specificity of the mammalian neutral metalloendopeptidase, endopeptidase 24.11, with that of the bacterial neutral metalloendopeptidase thermolysin. A series of synthetic oligopeptides which have previously been studied as substrates for thermolysin and used in computer modeling were examined as substrates for the mammalian enzyme. It was found that P1, P2, and P'3 subsite interactions in the mammalian enzyme, although similar to those found in thermolysin, are less restrictive spatially and are considerably less dependent on hydrophobic interactions. This difference was maximally expressed with the synthetic substrate dansyl-D-alanylglycylnitrophenylalanylglycine which is a substrate for the mammalian enzyme, but not for the bacterial enzyme. A comparison of substrates in the free acid form with their corresponding amides showed that binding to the mammalian enzyme is dependent in part on an ionic interaction between the substrate carboxylate group and the enzyme. Such an ionic interaction was not observed with the bacterial enzyme.

Amino Acid Sequence↗
PubMed1984

Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts.

Several proteases, i.e., pronase, a mixture of trypsin and chymotrypsin, and thermolysin were screened as potential surface probes of isolated intact pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Of these, only thermolysin met the criteria of a suitable probe. Thermolysin destroyed outer envelope polypeptides, but did not affect inner envelope polypeptides, envelope permeability properties or such chloroplast activities as metabolite transport and O(2) evolution.

Journal Article↗
PubMed2002

Reversible acetonitrile-induced inactivation/activation of thermolysin.

Thermolysin is catalytically inactive in mixtures of 10-15 % acetonitrile in aqueous buffer. Unexpectedly, dilution of the inactive enzyme with acetonitrile leads to complete recovery of the catalytic activity in a similar way to dilution with aqueous buffer. Circular dichroism and fluorescence studies of thermolysin in the same solvent mixtures reveal discontinuous changes in the overall secondary and tertiary protein structure that correlate well with the reversible differences in catalytic activity. The spectra on either side of the minimum activity point are different from each other, a fact indicating that the enzyme may be able to access two active conformations which are thermodynamically stable in different solvent environments.

Acetonitriles↗
PubMed1993

Modulation of a novel thermolysin-like metallo-endopeptidase activity during retinoic acid-induced differentiation of human neuroectodermal tumor cell lines.

Neuroectodermal tumours express hormones which are post-translationally processed and inactivated by the action of specific proteases and peptidases. The data reported here show the presence of a novel thermolysin-like metallo-endopeptidase activity in several human cell lines. The soluble fractions of neuroblastoma, melanoma and a glioblastoma tumour cell lines are able, with different degrees, to cleave the Ser12-Phe13 bond of a DVDERDVRGFAS decreases FLNH2 substrate. The inhibition pattern suggests a metallo-endopeptidase thermolysin-like character, with the involvement of thiol group(s), clearly distinct from neutral endopeptidase (NEP; EC 3.4.24.11). This metallo-endopeptidase activity is down regulated during retinoic acid(RA)-induced neuronal differentiation in the RA-sensitive SK-N-BE(2) cells but not in the RA-resistant BE(2)-M17 cells, suggesting that the down regulation is related to neuronal differentiation and not a direct effect of RA on the enzymatic activity.

Amino Acid Sequence↗