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PubMed1995

The essential dynamics of thermolysin: confirmation of the hinge-bending motion and comparison of simulations in vacuum and water.

Comparisons of the crystal structures of thermolysin and the thermolysin-like protease produced by B. cereus have recently led to the hypothesis that neutral proteases undergo a hinge-bending motion. We have investigated this hypothesis by analyzing molecular dynamics simulations of thermolysin in vacuum and water, using the essential dynamics method. This method is able to extract large concerted atomic motions of biological importance from a molecular dynamics trajectory. The analysis of the thermolysin trajectories indeed revealed a large rigid body hinge-bending motion of the N-terminal and C-terminal domains, similar to the motion hypothesized from the crystal structure comparisons. In addition, it appeared that the essential dynamics properties derived from the vacuum simulation were similar to those derived from the solvent simulation.

Computer Simulation↗
PubMed1995

Reaction of neprilysin (neutral endopeptidase) and thermolysin with cyclic peptides.

The reaction of neprilysin and thermolysin with a series of cyclic beta-turn peptides, varying in length from 6 to 14 residues, has been studied. All of the cyclic peptides bind to neprilysin with their affinity increasing from 113 microM for the 6-membered ring to 17 microM for the 14-membered ring. The 6-membered cyclic peptide was not hydrolyzed. However, kcat increased from 1.5 min-1 for the 8-membered cyclic peptide to 148 min-1 for the 14-membered cyclic peptide. With thermolysin binding of the 6- or 8-membered cyclic peptides was not detected. The Km values for the 10-, 12-, and 14-membered cyclic peptides were all in the 100 microM range. With thermolysin, kcat increased from 7 min-1 for the 10-membered cyclic peptide to 27,000 min-1 for the 14-membered cyclic peptide. Cyclic peptides were all cleaved at N-terminally directed sites. Modeling of the binding of a cyclic peptide, structurally similar to the 12-membered cyclic beta-turn peptide described above, into the active site of thermolysin shows that only half of the substrate makes contact with the enzyme and that only residues on one side of the peptide could fit into the active site. From these studies it is concluded that key factors which influence catalysis include not only peptide sequence, but the flexibility of the peptide and the orientation of the S'1 residue in a cyclic peptide.

Amino Acid Sequence↗
PubMed2005

Exploring the subsite-structure of vimelysin and thermolysin using FRETS-libraries.

Vimelysin is a metalloproteinase with high activity at low temperature and an unusual resistance to organic solvents. Substrate specificities of vimelysin and thermolysin were examined using FRETS-libraries, revealing a significant difference at the P3' position: vimelysin preferred acidic amino acid residues, whereas thermolysin preferred basic residues. Homology modeling of vimelysin suggests that oppositely charged residues in the S3' subsites (R215 in vimelysin and D213 in thermolysin) may be responsible for this specificity difference. This hypothesis was confirmed by examining the R215D mutant of vimelysin, which showed a substrate specificity profile intermediate between thermolysin and vimelysin.

Combinatorial Chemistry Techniques↗
PubMed2006

Effects of water-miscible solvents and polyhydroxy compounds on the structure and enzymatic activity of thermolysin.

The effect of organic solvents (n-propanol, isopropanol, dimethylformamide and dimethylsulfoxide) on the structure, activity and stability of thermolysin was the focus of this investigation. Results show the ability of the solvents to cause mixed inhibition of thermolysin, which was indicated by kinetic and structural studies (near-UV CD spectra and intrinsic fluorescence). Inhibitory effect of the solvents increased with increments in solvents logP. Thermoinactivation of thermolysin was studied at 80 degrees C in 50% of solvents and showed that with the increase in solvent hydrophobicity, thermal stability of the enzyme decreased. For the stabilization of thermolysin at high temperature, additives such as glycerol, sorbitol and trehalose were employed. In the presence of DMF with a relatively low logP, trehalose was shown to be a good stabilizer, whereas glycerol had a marked stabilization effect in the presence of n-propanol and isopropanol with a relatively high logP. Consequently, it was concluded that the stabilizing effect of additives can be correlated with the logP of solvents.

1-Propanol↗
PubMed1998

Differences in transition state stabilization between thermolysin (EC 3.4.24.27) and neprilysin (EC 3.4.24.11).

Important homologies in the topology of the catalytic site and the mechanism of action of thermolysin and neprilysin have been evidenced by site-directed mutagenesis. The determination of differences in transition state stabilization between these peptidases could facilitate the design of specific inhibitors. Thus, two residues of thermolysin which could be directly (Tyr157) or indirectly (Asp226) involved in the stabilization of the transition state and their putative counterparts in neprilysin (Tyr625 and Asp709) have been mutated. The results show that Tyr157 is important for thermolysin activity while Tyr625 has no functional role in neprilysin. Conversely, the mutation of Asp226 induced a slight perturbation of thermolysin activity while Asp709 in neprilysin seems crucial in neprilysin catalysis. Taken together these data seem to indicate differences in the transition state mode of stabilization in the two peptidases.

Amino Acid Sequence↗
PubMed1994

Kinetics of hydrolysis of dansyl peptide substrates by thermolysin: analysis of fluorescence changes and determination of steady-state kinetic parameters.

The stopped-flow fluorescence technique has been used to study the hydrolysis of 10 dansyl peptides by thermolysin. The origin of the fluorescence changes observed during the reactions has been investigated in detail. Depending on the substrate and the excitation wavelength, the dansyl fluorescence changes observed arise either from energy transfer (maximal at lambda ex = 230 and 280 nm) between Trp residues of thermolysin and the dansyl group of the substrate in enzyme-substrate (ES) complexes or from both sources. These excitation (maximal at lambda ex = 245 and 340 nm) of the free substrate and product, or from both sources. These two types of fluorescence signals reflect the concentrations of ESi and free substrate, respectively. Both types of fluorescence changes have been used to monitor the reaction progress, and different mathematical formalisms have been used to determine the kinetic parameters for the reactions with results that are in good agreement. The efficiency of Trp quenching by a series of five dansyl tripeptides is shown to be related to the fractional saturation of enzyme and follows the KM-1 values for the substrates. The quenching efficiency for a dansyl tetrapeptide is weaker due to the greater distance between the dansyl group and the Trp-115 donor in thermolysin. On the basis of these studies, substrates capable of supporting more detailed kinetic studies of thermolysin have been identified.

Amino Acid Sequence↗
PubMed1983

Structural analysis of the inhibition of thermolysin by an active-site-directed irreversible inhibitor.

The mode of binding of the irreversible thermolysin inhibitor ClCH2CO-DL-(N-OH)Leu-OCH3 [Rasnick, D., & Powers, J.C. (1978) Biochemistry 17, 4363-4369] has been determined by X-ray crystallography at a resolution of 2.3 A and the structure of the covalent complex refined to give a crystallographic residual of 17.0%. This is the first such structural study of an active-site-directed covalent complex of a zinc protease. As anticipated by Rasnick and Powers, the inhibitor alkylates Glu-143 in the thermolysin active site, and the hydroxamic acid moiety coordinates the zinc ion. The formation of the covalent complex is associated with a significant shift in a segment of the polypeptide backbone in the vicinity of the active site. This conformational adjustment appears to be necessary to relieve steric hindrance which would otherwise prevent alkylation of Glu-143. It is suggested that this steric hindrance, which occurs for thermolysin but would not be expected for carboxypeptidase A, accounts for the previously inexplicable difference in reactivity of these two metalloproteases toward N-haloacetyl amino acids. The relevance of this steric hindrance to the mechanism of catalysis is discussed. In agreement with previous results [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506-2516], it appears that steric hindrance prevents the direct attack of Glu-143 on the carbonyl carbon of an extended substrate, therefore ruling out the anhydride pathway in thermolysin-catalyzed hydrolysis of polypeptide substrates and their ester analogues.

Binding Sites↗
PubMed1984

Independent folding of the carboxyl-terminal fragment 228-316 of thermolysin.

The COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1984

Binding of N-carboxymethyl dipeptide inhibitors to thermolysin determined by X-ray crystallography: a novel class of transition-state analogues for zinc peptidases.

The mode of binding of the specific thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan (KI approximately 5 X 10(-8) M) [Maycock, A. L., DeSousa, D. M., Payne, L. G., ten Broeke, J., Wu, M. T., & Patchett, A. A. (1981) Biochem. Biophys. Res. Commun. 102, 963-969] has been determined by X-ray crystallography and refined to an R value of 17.1% at 1.9-A resolution. The inhibitor binds to thermolysin with both oxygens of the N-carboxymethyl group liganded to the zinc to give overall pentacoordination of the metal. The bidentate ligation of the inhibitor differs from the monodentate binding seen previously for carboxylate-zinc interactions in thermolysin and is closer to the bidentate geometry observed for the binding of hydroxamates [Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912-6920]. The geometry of the inhibitor and its interactions with the protein have a number of elements in common with the presumed transition state formed during peptide hydrolysis. The observed zinc ligation supports the previous suggestion that a pentacoordinate intermediate participates in the mechanism of catalysis. However, the alpha-amino nitrogen of the inhibitor is close to Glu-143, suggesting that this residue might accept a proton from an attacking water molecule (as proposed before) and subsequently donate this proton to the leaving nitrogen. By analogy with thermolysin, it is proposed that a related mechanism should be considered for peptide cleavage by carboxypeptidase A.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1988

Primary structure homologies between two zinc metallopeptidases, the neutral endopeptidase 24.11 ("enkephalinase") and thermolysin, through clustering analysis.

Analogies in the sequences of two related zinc metallopeptidases, the bacterial thermolysin (316 amino acids) and the recently cloned neutral endopeptidase 24.11 ("enkephalinase", 749 amino acids), have been demonstrated by a hydrophobic cluster analysis method derived from the Lim theory. Two sequence alignments are proposed for the entire primary structure of thermolysin and the C-terminal part of endopeptidase 24.11. Except for an arginine residue, all the amino acids involved in the active site of thermolysin have been retrieved in both models of endopeptidase 24.11 within conserved clustered structures. The first model is characterized by a deletion of the Ca2+-binding coil present in thermolysin and the second by replacement of this coil by two alpha-helices. In both models an Arg residue can be located in the active site of the neutral endopeptidase.

Amino Acid Sequence↗
PubMed1990

Inhibition of thermolysin by phosphonamidate transition-state analogues: measurement of 31P-15N bond lengths and chemical shifts in two enzyme-inhibitor complexes by solid-state nuclear magnetic resonance.

31P and 15N chemical shifts and 31P-15N bond lengths have been measured with solid-state NMR techniques in two inhibitors of thermolysin, carbobenzoxy-Glyp-L-Leu-L-Ala (ZGpLA) and carbobenzoxy-L-Phep-L-Leu-L-Ala (ZFpLA), both as free lithium salts and when bound to the enzyme. Binding of both inhibitors to thermolysin results in large changes in the 31P chemical shifts. These changes are more dramatic for the tighter binding inhibitor ZFpLA, where a approximately 20 ppm downfield movement of the 31P isotropic chemical shift (sigma iso) is observed. This shift is due to changes in the shift tensor elements sigma 11 and sigma 22, while sigma 33 remains essentially constant. We observed a similar pattern for ZGpLA, but only a approximately 5 ppm change occurs in sigma iso. The changes in the 15N chemical shifts for both inhibitors are small upon binding, amounting to downfield shifts of 2 and 4 ppm for ZGpLA and ZFpLA, respectively. This indicates that there are no changes in the protonation state of the 15N in either the ZFpLA- or the ZGpLA-thermolysin complex. NMR distance measurements yield a P-N bond length rP-N = 1.68 +/- 0.03 A for the tight binding inhibitor ZFpLA both in its free lithium salt form and in its thermolysin-ZFpLA complex, a distance that is much shorter than the 1.90-A distance reported by X-ray crystallography studies [Holden et al. (1987) Biochemistry 26, 8542-8553].(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Structural determinants of the stability of thermolysin-like proteinases.

Thermolysin is a member of a family of homologous proteinases which differ in their resistance to thermally induced unfolding and subsequent autolytic degradation. Site-directed mutagenesis studies of the thermolysin-like proteinase (TLP) from Bacillus stearothermophilus (TLP-ste) show that its reduced resistance to thermally induced autolysis, as compared to thermolysin, is due to only some of the 44 naturally occurring amino-acid differences between them. In fact TLP-ste becomes more resistant than thermolysin by mutation of just a few of these amino-acids. The crucial differences are all localized to a solvent-exposed region in the N-terminal domain of TLP-ste.

Amino Acid Sequence↗
PubMed1985

Purification of human urinary prokallikrein. Identification of the site of activation by the metalloproteinase thermolysin.

Human urinary active kallikrein and prokallikrein were separated on DEAE-cellulose and octyl-Sepharose columns and both purified to homogeneity by affinity chromatography, gel filtration and hydrophobic h.p.l.c. Prokallikrein was monitored during purification by trypsin activation followed by determination of both amidase and kininogenase activity. After trypsin activation, purified prokallikrein had a specific kininogenase activity of 39.4 micrograms of bradykinin equivalent/min per mg and amidase activity of 16.5 mumol/min per mg with D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin. Purified active kallikrein had a specific activity of 47 micrograms of bradykinin/min per mg. The molecular mass of prokallikrein was 48 kDa on electrophoresis and 53 kDa on gel filtration whereas active kallikrein gave values of 46 kDa and 53 kDa respectively. Antisera to active and prokallikrein were obtained. In double immunodiffusion and immunoelectrophoresis, antiserum to active kallikrein reacted with active and pro-kallikrein. Antiserum to prokallikrein contained antibodies to determinants not found in active kallikrein, presumably due to the presence of the activation peptide in the proenzyme. Human prokallikrein can be activated by thermolysin, trypsin and human plasma kallikrein. Activation of 50% of the prokallikrein (1.35 microM) was achieved in 30 min with 25 nM-thermolysin, 78 nM-trypsin or 180 nM-human plasma kallikrein. Thus thermolysin was the most effective activator. Thermolysin activated prokallikrein by releasing active kallikrein with N-terminal Ile1-Val2. Thus human tissue (glandular) prokallikrein can be activated by two types of enzymes: serine proteinases, which cleave at the C-terminus of basic amino acids, and by a metalloproteinase that cleaves at the N-terminus of hydrophobic amino acids.

Amino Acids↗
PubMed2001

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.

Amino Acid Substitution↗
PubMed1997

Effects of pH, temperature, and alcohols on the remarkable activation of thermolysin by salts.

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl] (FA)-dipeptide amides and N-carbobenzoxyl-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced by high concentrations (1-5 M) of neutral salts. The activation is due to an increase in the molecular activity, k(cat), while the Michaelis constant, K(m), is not affected by the addition of NaCl. In the present study, the effect of NaCl on the thermolysin-catalyzed hydrolysis of FA-glycyl-L-leucine amide (FAGLA) has been examined by changing the pH and temperature, and by adding alcohols to the reaction mixture. The enzyme activity, expressed by k(cat)/K(m), is pH-dependent, being controlled by two functional residues with pK(a) values of 5.4 and 7.8 in the absence of NaCl. The acidic pK(a) is shifted from 5.4 to 6.7 by the addition of 4 M NaCl, while the basic one is not changed. The degree of activation at a given concentration of NaCl is pH dependent in a bell-shaped manner with the optimum pH around 7. Although the activity increases in both the presence and absence of NaCl with increasing temperature from 5 to 35 degrees C, the degree of activation decreases. Alcohols inhibit thermolysin, and the degree of activation decreases with increasing alcohol concentration. The degree of activation tends to increase with increasing dielectric constant of the medium, although it varies considerably depending on the species of alcohol. Electrostatic interactions on the surface and at the active site of thermolysin are suggested to play a significant role in the remarkable activation by salts.

Acrylates↗
PubMed1983

Studies on the chemical modification of tryptophan residues in thermolysin and in talopeptin (MKI) with N-bromosuccinimide.

Tryptophan residues in thermolysin (3 Trp/molecule) and in its specific inhibitor, talopeptin (1 Trp/molecule), were modified with N-bromosuccinimide (NBS). The decrease in the absorption at 280 nm and the fluorescence intensity above 310 nm (excited at 280 nm) accompanying the modification were followed by the stopped-flow method as a function of time. When the sole tryptophan residue of talopeptin was modified with NBS, its inhibitory activity against thermolysin was almost completely destroyed. For thermolysin, the decrease in molar absorptivity corresponds to the modification of one of its three tryptophan residues, and the enzyme activity does not decrease significantly with the modification (remaining activity was 96% at [NBS]/[E] = 6). The results obtained for the modification of EI complex suggested that the formation of EI complex remarkably reduces the rate constant for the modification of the tryptophan residue in talopeptin, but does not affect that of the tryptophan residue(s) in thermolysin.

Bromosuccinimide↗
PubMed1990

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin.

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val- Gly-Val-Lys, corresponding to sequence 296-316 of thermolysin and thus encompassing the COOH-terminal helical segment 301-312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296-316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296-307, 308-316, 296-302, and 303-316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296-316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301-312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296-316 attains in 90% aqueous trifluoroethanol the same percentage (approximately 58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296-316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule.

Alcohols↗
PubMed1976

Role of calcium ions in the thermostability of thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease.

The stabilizing effect of calcium ions on thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease has been investigated. Calcium and zinc ions were removed from the proteases by gel filtration over Sephadex G-25 equilibrated with metal chelating agents. Using these enzymes with different metal content, heat inactivation kinetics were studied at various temperatures. Removal of calcium ions caused a sharp decrease in thermostability and diminished the values of the activation enthalpy (deltaH*) and entropy (deltaS*) for heat inactivation. There was little difference in stability between thermolysin containing 0.3 g-atom/mol and B. subtilis neutral protease containing 1.4 g-atoms/mol. Calcium binding isotherms of the proteases were obtained by equilibrium gel chromatography with various concentrations of free calcium ions. Thermolysin had four independent calcium binding sites with an identical intrinsic binding constant (K) of 2.0 X 10(4) M-1. B. subtilis neutral protease had four independent sites. The K value for three sites was 1.1 X 10(5) M-1 and the binding constant for the other site was 1.5 X 10(3) M-1. There was little difference in total free energy change for calcium binding between these proteases. From these results it is concluded that the stabilizing effect of calcium on these enzymes is almost equal, and the extra thermal stability of thermolysin is likely to come from its polypeptide chain structure.

Bacillus subtilis↗