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 55 records · Page 3Linked to original sources
PubMed1985

Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure.

The COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin.

Amino Acid Sequence↗
PubMed1988

Crystal structure of neutral protease from Bacillus cereus refined at 3.0 A resolution and comparison with the homologous but more thermostable enzyme thermolysin.

Neutral protease from Bacillus cereus exhibits a 73% amino acid sequence homology to thermolysin, for which an accurate crystal structure exists. The B. cereus enzyme is, however, markedly less thermostable. The neutral protease was crystallized and diffraction data to 3.0 A resolution were recorded by oscillation photography. The crystal structure was solved by molecular replacement methods using thermolysin as a trial molecule. The solution was improved by rigid-body refinement and model rebuilding into electron density omit-maps. The atomic co-ordinates were refined to R = 21.7% at 3.0 A resolution. Comparison of the resultant model with the thermolysin structure shows that the two enzymes are very similar with a root-mean-square deviation between equivalent C alpha-atoms of 0.88 A. The gamma-turn found in thermolysin is transformed into a beta-turn in the neutral protease by the insertion of a glycine residue. There appear to be no contributions to the enhanced thermostability of thermolysin from additional salt bridges, whereas contributions in the form of extra hydrogen bonding interactions could be important. Other factors that may affect thermostability include the two glycine to alanine exchanges and perturbations in the environment of the double calcium site.

Amino Acid Sequence↗
PubMed1988

Structure and stability of thermophilic enzymes. Studies on thermolysin.

The molecular mechanisms responsible for the unusual stability of enzymes isolated from thermophilic microorganisms are much more complex and subtle than was originally thought. In particular, a general mechanism cannot be proposed, since individual enzymes can be stabilized by specific molecular interactions and forces. The results of studies on thermophilic enzymes obtained in recent years in our laboratory will be summarized, with particular emphasis being placed on those obtained with thermolysin, a stable metalloendopeptidase isolated from Bacillus thermoproteolyticus. Fragmentation of thermolysin by limited proteolysis by added protease (subtilisin) or autolysis mediated by heat or the ion-chelating agent EDTA leads to quite selective peptide bond fissions, allowing isolation of 'nicked' thermolysin species. Correlation of the sites of proteolytic cleavage with the known three-dimensional structure of thermolysin allowed us to infer some of the key characteristics of the structure, folding, dynamics and stability of the thermolysin molecule. The potential utility of these and other studies on thermophilic enzymes in devising strategies for enhancing the stability of mesophilic enzymes using genetic engineering techniques is discussed.

Amino Acid Sequence↗
PubMed1994

NMR solution structure of the C-terminal fragment 255-316 of thermolysin: a dimer formed by subunits having the native structure.

The solution structure of the C-terminal fragment 255-316 of thermolysin has been determined by two-dimensional proton NMR methods. For this disulfide-free fragment there was a previous proposal according to which it would fold into a stable helical structure forming a dimer at concentrations above 0.06 mM. A complete assignment of the proton NMR resonances of the backbone and amino acid side chains of the fragment was first performed using standard sequential assignment methods. On the basis of 729 distance constraints derived from unambiguously assigned nuclear Overhauser effect (NOE) proton connectivities, the three-dimensional structure of a monomeric unit was then determined by using distance geometry and restrained molecular dynamic methods. The globular structure of fragment 255-316 of thermolysin in solution, composed of three helices, is largely coincident with that of the corresponding region in the crystallographic structure of intact thermolysin [Holmes, M. A., & Matthews, B. W. (1982) J. Mol. Biol. 160, 623-639]. This fact allowed identification as intersubunit of up to 52 NOE cross correlations, which were used to dock the two subunits into a symmetric dimer structure. The obtained dimeric structure served as the starting structure in a final restrained molecular dynamic calculation subjected to a total of 1562 distance constraints. In the resulting dimeric structure, the interface between the two subunits, of a marked hydrophobic character, coincides topologically with the one between the 255-316 fragment and the rest of the protein in the intact enzyme. The present work decisively shows that the thermolysin fragment 255-316 can attain a stable and nativelike structure independently of the rest of the polypeptide chain. Considering that the thermolysin molecule is constituted of two structural domains of equal size (residues 1-157 and 158-316), the results of this study show that autonomously folding units can be substantially smaller than entire domains.

Amino Acid Sequence↗
PubMed1997

NMR solution structure of the 205-316 C-terminal fragment of thermolysin. An example of dimerization coupled to partial unfolding.

The solution structure of the C-terminal fragment 205-316 of thermolysin has been determined by 1H-NMR methods. The fragment forms a dimer in which each subunit has two different regions: the largely disordered N-terminal segment 205-260 and the structurally well-defined segment 261-316. The structured part of each subunit is composed of three helices and is largely coincident with the corresponding region in the solution structure of the dimer formed by the shorter fragment 255-316, which in turn coincides with the crystallographic structure of intact thermolysin. As with the fragment 255-316, the subunit interface is highly hydrophobic and coincides topologically with the one between the segment 255-316 and the rest of the protein in the intact enzyme. A fourth helix (residues 235-246), present in the segment 205-316 of native thermolysin, is mostly disordered in the dimer formed by the fragment 205-316. The location of the fourth helix in the native structure of intact thermolysin does not allow the formation of the dimer interface observed in the solution structure of the fragment 255-316. Under the NMR conditions, dimer formation is energetically more favorable than the dissociated monomers. The latter, based on calorimetric data, was proposed to have partial structure in the region 205-254 as in native thermolysin. Thus, it appears that the assembly of the dimer would require an initial unfolding in the region 205-254 of the monomer.

Amino Acid Sequence↗
PubMed1997

A theoretical investigation of tight-binding thermolysin inhibitors.

A tight-binding thermolysin inhibitor, Cbz-Phe-psi[PO2NH]-Leu-Ala (ZFpLA, Ki = 0.068 nM), and its analogs, ZRp(O)LA (R = Ala, Leu or Phe) have been studied using the finite difference solution to the linearized Poisson-Boltzmann equation (FDPB) and solvation entropy correction (SEC). The binding energy difference between conformationally different thermolysin inhibitors ZFpLA and ZGpLL is estimated using three approaches. Two of approaches use the X-ray structures of ZFpLA-thermolysin and ZGpLL-thermolysin structures. The third one uses both X-ray structures to calculate binding energy differences from ZFpLA and ZGpLL to a hypothetical intermediate MepLA. All the results are qualitatively correct with one closely reproducing the experimental value. The enhancement of the ZFpLA binding is attributed largely to the solvation entropy or "hydrophobic force". The binding mode of the ZGpLR N-terminal moiety appears to be electrostatically unfavorable. Reducing the polarity of that moiety is predicted to enhance binding affinity. The binding trends due to the hydrophobic variation of ZRp(O)LA are calculated within 1 kcal/mol of the experimental values. Increasing lipophilicity of a ligand favors the binding due to the difference of surface area change between the free state and the bound state. The analysis of energetic components shows that these trends are not specific for the binding of phosphorus-containing inhibitors but are generally true for protein-ligand interactions. The electrostatic calculation does not support the involvement of the second protonation of ZFpLA in binding. Therefore, reexamining the second protonation of ZFpLA or seeking further experimental support seems appropriate. The structural sensitivity of the FDPB calculation was assessed by using ligand and receptor structures from different X-ray studies of thermolysin. The small deviations (< 0.3 A) in the receptor structures do not cause significant changes in electrostatic binding energy if there is no structural change in modified regions.

Binding Sites↗
PubMed1977

A fluorimetric study of the role of calcium ions in the stability of thermolysin.

1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn(2+) ion and the four Ca(2+) ions/molecule from the enzyme. 2. At 25 degrees C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N'-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80 degrees C, for the apoenzyme a thermal transition is observed near 48 degrees C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn(2+) ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca(2+) ions, which have a bridging function within this disulphide-free protein molecule.

Calcium↗
PubMed1994

Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin.

We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn37 and Gln119, originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.

Amino Acid Sequence↗
PubMed2002

Thermolysin-linearized microcin J25 retains the structured core of the native macrocyclic peptide and displays antimicrobial activity.

Microcin J25 (MccJ25) is the single macrocyclic antimicrobial peptide belonging to the ribosomally synthesized class of microcins that are secreted by Enterobacteriaceae. It showed potent antibacterial activity against several Salmonella and Escherichia strains and exhibited a compact three-dimensional structure [Blond et al. (2001) Eur. J. Biochem., 268, 2124-2133]. The molecular mechanisms involved in the biosynthesis, folding and mode of action of MccJ25 are still unknown. We have investigated the structure and the antimicrobial activity of thermolysin-linearized MccJ25 (MccJ25-L1-21: VGIGTPISFY10GGGAGHVPEY20F), as well as two synthetic analogs, sMccJ25-L1-21 (sequence of the thermolysin-cleaved MccJ25) and sMccJ25-L12-11 (C-terminal sequence of the MccJ25 precursor: G12GAGHVPEYF21V1GIGTPISFYG11). The three-dimensional solution structure of MccJ25-L1-21, determined by two-dimensional NMR, consists of a boot-shaped hairpin-like well-defined 8-19 region flanked by disordered N and C termini. This structure is remarkably similar to that of cyclic MccJ25, and includes a short double-stranded antiparallel beta-sheet (8-10/17-19) perpendicular to a loop (Gly11-His16). The thermolysin-linearized MccJ25-L1-21 had antibacterial activity against E. coli and S. enteritidis strains, while both synthetic analogues lacked activity and organized structure. We show that the 8-10/17-19 beta-sheet, as well as the Gly11-His16 loop are required for moderate antibacterial activity and that the Phe21-Pro6 loop and the MccJ25 macrocyclic backbone are necessary for complete antibacterial activity. We also reveal a highly stable 8-19 structured core present in both the native MccJ25 and the thermolysin-linearized peptide, which is maintained under thermolysin treatment and resists highly denaturing conditions.

Amino Acid Sequence↗
PubMed1994

A spectrophotometric study on the interaction of thermolysin with chloride and bromide ions, and the state of tryptophyl residue 115.

The activity of thermolysin is greatly enhanced in the presence of high concentrations of neutral salts [Holmquist, B. and Vallee, B.L. (1976) Biochemistry 15, 101-107; Inouye, K. (1992) J. Biochem. 112, 335-340]. NaBr and NaCl are the most effective for the activation. An absorption difference spectrum with a peak around 293 nm, which is characteristic of the red-shift of a tryptophyl residue caused by charge effects, was observed on mixing of thermolysin with NaCl. As the peak disappeared in the presence of competitive inhibitors of the enzyme (phosphoramidon and zincov), it was considered to be derived from a tryptophyl residue (Trp 115) located in the active site of the enzyme. On the other hand, this peak was not observed on the mixing of thermolysin and NaBr, indicating that the slight difference in size between chloride and bromide ions is critical for the interaction with the tryptophyl residue. NaCl and NaBr exhibit comparable effects on the activation of thermolysin regardless of the considerable discrepancy in their effects on the absorptivity difference around 293 nm. This suggests that the interaction of salts with Trp 115 is not necessarily correlated with the activation of thermolysin.

Binding Sites↗
PubMed1983

Domain characteristics of the cyanogen bromide fragment 121-316 of thermolysin.

The molecule of thermolysin was shown by X-ray crystallography to be composed of two structural domains of equal size comprising residues 1-157 and 158-316. In order to explore the possibility that these domains correspond to globular fragments able to refold autonomously, we have investigated the conformational and stability properties of fragment 121-316, which was obtained by limited chemical cleavage of thermolysin with cyanogen bromide. As judged by far-ultraviolet circular dichroism measurements, in aqueous solution under neutral conditions the fragment maintains a relative amount of helical structure which is comparable to that exhibited by the corresponding region in native thermolysin. The secondary structure attained by the fragment appears remarkably stable to the denaturing action of heat. By measuring the temperature dependence of the dichroic signal at 220 nm a Tm near 74 degrees was obtained. Immunodiffusion analyses indicated that the fragment recognizes and precipitates antibodies raised in rabbits using native thermolysin as immunogen. The overall conformational and immunochemical data indicate that fragment 121-316 of thermolysin is able to refold into a stable structure of native-like characteristics independently of the rest of the molecule. The results of this study complement those previously reported for fragment 206-316 (Vita, C., Fontana, A., Seeman, J.R. & Chaiken, I.M. (1979) Biochemistry 18, 3023-3031).

Amino Acids↗
PubMed1981

Limited proteolysis of porcine-muscle lactic dehydrogenase by thermolysin during reconstitution yields dimers.

Reassociation of lactic dehydrogenase from porcine skeletal muscle at 10 degrees C was studied by limited proteolysis during reconstitution, applying thermolysin as proteolytic enzyme. Proteolysis was achieved by short incubation of the reassociating enzyme with the protease in a ratio lactic dehydrogenase:thermolysin = 10:1 (0.1 M sodium phosphate buffer pH 7.6 plus 1 mM dithioerythritol). Thermolysin was found to satisfy all the obligatory requirements for proteolytic studies of reconstituting enzymes: (a) the protease is essentially inactive towards native lactic dehydrogenase; (b) it splits intermediates of reconstitution at a high rate; (c) it can be inactivated instantaneously by the addition of 10 mM EDTA. As demonstrated by gel chromatography, thermolysin digestion stabilizes dimers of porcine muscle lactic dehydrogenase as intermediates of reconstitution. These 'dimers' consist of virtually intact polypeptide chains of molecular weight about 35000, apart from nicked subunits with molecular weights of 18000 and 12000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analyzing the kinetics of reassociation by thermolysin digestion at various times during reconstitution, and subsequent gel filtration, the dimer replaced by tetramer transition is found to be rate-limiting for both reassociation and reactivation. This result corroborates a previously proposed model for the reconstitution of lactic dehydrogenase, based on either cross-linking experiments [R. Hermann et al. (1979) Nature (Lond.) 277, 243--245], or the reassembly of dimeric intermediates of dissociation [R. Jaenicke et al. (1981) Eur. J. Biochem. 114, 525--531].

Animals↗
PubMed1986

Domain unfolding and the stability of thermolysin in guanidine hydrochloride.

Equilibrium and kinetic studies of the unfolding and autolysis of the two domain protein thermolysin in guanidine hydrochloride are described. Enzyme activity, circular dichroism, fluorescence, sedimentation, size exclusion chromatography, and viscosity measurements were used to monitor conformational transitions and characterize the native and denatured states. The observation of biphasic transitions for the unfolding of apothermolysin and the spectroscopic changes associated with each phase of the overall unfolding process suggest unfolding of the N-terminal domain at less than 1 M guanidine hydrochloride, followed by the unfolding of the C-terminal domain, with the transition midpoint at 3 M guanidine hydrochloride. The refolding of the C-terminal domain is reversible; however, refolding of the N-terminal domain could not be demonstrated owing to protein aggregation. A quantitative analysis of the two transitions suggest that the unfolding of the two structural domains of thermolysin is not completely independent. Attempts to measure the unfolding of holothermolysin were hampered by autolysis. However, it was possible to show that at least three calcium ions serve to stabilize thermolysin against autolysis or unfolding in guanidine hydrochloride. Similar stabilization was observed for thermolysin with a single terbium ion bound at calcium site S(1). This result is consistent with our earlier findings, which suggest that calcium bound at sites S(1)-S(2) are located at a critical point on the unfolding pathway of thermolysin and serve to act as an interdomain lock.

Apoenzymes↗
PubMed1986

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase.

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.

Amino Acid Sequence↗
PubMed1980

Thermolysin and Bacillus subtilis neutral protease. Conformation and stability of two homologous neutral metalloendopeptidases.

A comparative study on thermolysin from Bacillus thermoproteolyticus and neutral protease from Bacillus subtilis involving (far- and near-ultraviolet circular dichroism (CD) and immunological techniques is reported. These enzymes are homologous metalloendopeptidases, having similar size, kinetic behaviour, substrate specificity and susceptibility to inhibitors. The far-ultraviolet CD spectrum of each protein shows a minimum at 208 and a shoulder near 220 nm; differences in the extent of ellipticity, however, have been observed. Estimates of secondary structure obtained by quantitation of the far-ultraviolet CD spectra indicated a higher helicity of neutral protease relative to thermolysin. In the presence of ethylenediaminetetraacetic acid, which removes calcium and the functional zinc ion from the metalloenzymes, neutral protease is immediately denatured, whereas thermolysin maintains a globular structure, although thermolabile. On the other hand, the zinc-specific chelating agent tetraethylenepentamine does not have measurable effects on the conformation and conformational stability of either protein. Marked higher stability to temperature and guanidine hydrochloride were observed for thermolysin as compared with neutral protease, as indicated by monitoring conformational transitions with CD measurements at 220 nm. Antisera prepared in rabbits using thermolysin as immunogen do not cross-react with neutral protease, indicating differences of surface structure between the two proteins. On the basis and limitations of the techniques employed, it is proposed that the two sequentially and functionally homologous metalloendopeptidases may have similar conformations at specific regions (active and binding sites, at least) of the polypeptide chain essential for biological function, while some variability in the structure of other regions may be tolerated.

Amino Acids↗
PubMed2000

Media selection for refolding of thermolysin by use of immobilized preparation.

Selection of the most effective medium for the correct refolding of thermolysin was performed. Thermolysin that had been denatured with 6 M guanidinium chloride at pH 2.0 could not be recovered to its activity larger than ca. 10% even when the denaturant was diluted with a conventional buffer solution. The amount of activity recovered by this method decreased with time. The recovered activity was ca. 20% at most where 1 M calcium chloride or 1.6 M calcium acetate was employed as the refolding medium instead of the conventional buffer solution. In this case also, the activity decreased with time. Not only the low recovered activity or yield, but also the elimination of the activity once recovered, was probably mainly due to the intermolecular interactions between protein molecules such as autolysis and aggregation. In order to exclude the influence of the intermolecular interactions and to select the effective media for the correct refolding of thermolysin, immobilized thermolysin was prepared using agarose gel. Employment of the immobilized preparation made it possible to quantitatively determine the refolding of thermolysin and results revealed that the salts of organic acid, such as potassium acetate and sodium acetate, were excellent media for refolding. The immobilization was confirmed to be available for the selection of protein refolding media and indispensable, especially in the case of proteases. Since these results were partly similar to those obtained in the case of subtilisin reported previously, results of both cases were compared.

Journal Article↗
PubMed1995

Structural analysis of zinc substitutions in the active site of thermolysin.

Native thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active-site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601-4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal-substituted thermolysins at 1.7-1.9 A resolution. The structure of the Co(2+)-substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt-relative to the zinc-substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd(2+)- and Mn(2+)-substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side-chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side-chain movements are similar to adjustments that have been observed previously in association with the "hinge-bending" motion that is presumed to occur during catalysis by the zinc endoproteases. In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 A of the zinc bound to native thermolysin, explaining the inhibitory effect.

Amino Acid Sequence↗
PubMed1997

Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol.

We have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE-resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124-residue chain of RNase in its TFE state (20-42 degrees C, 6-24 h) at peptide bond Asn 34-Leu 35, followed by a slower cleavage at peptide bond Thr 45-Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1-34 and 35-124) (RNase Th1) or three (1-34, 35-45 and 46-124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Th1 maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-delta Tm 16 degrees C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Th1 is closely similar to that of the intact protein was confirmed unambiguously by two-dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30-41, i.e., at residues nearby the cleaved Asn 34-Leu 35 peptide bond. RNase Th1 retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 31-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a beta-strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Th1 and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE-state that likely dictate the selective proteolysis phenomenon by thermolysin.

Animals↗