Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tetanus neurotoxin (TeNT) blocks neuroexocytosis via a zinc-endopeptidase activity highly specific for vescicle-associated membrane protein(VAMP)/synaptobrevin. TeNT is the prototype of clostridial neurotoxins, a new family of metalloproteinases. They consist of three domains and the proteolytic activity is displayed by the 50-kDa light chain (L chain). The L chain was isolated here in the native state from bacterial filtrates of Clostridium tetani and its structure was studied via circular dichroism (CD) and fluorescence spectroscopy. The secondary structure content (27% alpha-helix and 43% beta-sheet), estimated by far-ultraviolet CD measurements, was in reasonable agreement with that obtained by standard predictive methods (25% alpha-helix and 49% beta-sheet). Moreover, the hypothetical zinc-binding motif, encompassing residues His-Glu-Leu-Ile-His, was correctly predicted to be in alpha-helical conformation, as also expected on the basis of the geometrical requirements for a correct coordination of the zinc ion. Both near-ultraviolet CD and fluorescence data strongly suggest that the single Trp43 residue is buried and constrained in a hydrophobic environment, likely distant from the zinc ion located in the active-site cleft. The contribution of the bound zinc ion to the overall conformation of TeNT L chain was investigated by different and complementary techniques, including spectroscopic (far- and near-ultraviolet CD, fluorescence, second derivative absorption spectroscopy) as well as proteolytic probes. The results indicate that the zinc ion plays little, if any, role in determining the structural properties of the L chain molecule. Similarly, the metal-free apo-enzyme and the holo-protein share common stability features evaluated in respect to different physico-chemical parameters (pH, temperature and urea concentration). These results parallel those obtained on thermolysin, a zinc-dependent neutral endoprotease from Bacillus thermoproteolyticus, where both conformational and stability properties are unchanged upon zinc removal.
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PMID:Structural studies on the zinc-endopeptidase light chain of tetanus neurotoxin. 774 50

The pattern and kinetics of partial proteolysis of Arthrobacter D-xylose isomerase tetramer was studied in order to determine the flexibility of surface loops that may control its stability. It was completely resistant to trypsin, chymotrypsin and elastase at 37 degrees C, but thermolysin cleaved specifically and quantitatively at Thr-347-Leu-348 between helices 10 and 11 to remove 47 residues from the C-terminus of each 43.3 kDa subunit. At high temperatures, helices 9 and 10 were removed from each 38 kDa subunit to give a 36 kDa tetramer. The kinetics of nicking by thermolysin indicated that the Thr-347-Leu-348 loop is locked at low temperatures, but 'melts' at 25 degrees C and is fully flexible above 34 degrees C. The flexibility appears to be associated with binding of Ca2+ ions at the active site, since Co2+, Mg2+ and xylitol protect in proportion to their ability to displace Ca2+. The missing C-terminal helices make many intersubunit contacts that appear in the structure to stabilize the tetramer, but the properties of the purified nicked proteins are almost indistinguishable from the native enzyme. Both the 38 kDa tetramer and the 36 kDa tetramer are identically active and dissociate similarly in urea or SDS to fully active dimers, but the nicked dimers are slightly less stable to urea at 62 degrees C. In the Mg2+ form the thermostability of the 38 kDa tetramer is identical with that of the native enzyme, but the 36 kDa tetramer has a slightly lower 'melting point' (70 degrees C versus 80 degrees C), which may be due to unravelling from the end of helix 8. Since elimination of all the C-terminal helices and many intersubunit contacts has so little effect, one can conclude that the 'weak point' that controls the protein's thermostability lies within the N-terminal beta-barrel domain.
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PMID:Arthrobacter D-xylose isomerase: partial proteolysis with thermolysin. 842 59

The unfolding and refolding rates of the heme-and Ca2+ -containing Coprinus cinereus peroxidase (CIP) have been measured at pH 12.1, in 4 M urea, and at 61.2 degrees C. The change in peroxidase activity paralleled the change in the Soret band absorbance of the heme group. The unfolding rate constant (ku), was determined independently in thermolysin digestion and EDTA experiments at 59.4 degrees C. Both gave ku values of 1.5 ms-1, and also showed that the presence of 4 mM EDTA made CIP unfolding practically irreversible. Unfolding and refolding rates could therefore be determined under identical conditions of denaturation having either EDTA or Ca2+ in excess. The refolding rates at high pH and in 4 M urea were measured by adding Ca2+ to the unfolded CIP, whereas refolding at 61.2 degrees C was evaluated by comparing the unfolding carried out under reversible (excess of Ca2+) and irreversible conditions (excess EDTA). The activation energies for the unfolding at 61.2 degrees C are approximately delta G++(u) 100, T delta S++(u) 200, and delta H++(u) 300 kJ/mol. Five different additives, glycerol, EtOH, Na2SO4, guanidinium chloride (GdmCl), and NaCl, all at 100 mM, were used as probes to evaluate the mechanism of base, urea, and heat on unfolding and refolding. Salts destabilized CIP at high pH, primarily by enhancing the unfolding rate but also by decreasing the refolding rate. Glycerol had the reverse effects and thus stabilized CIP at high pH. The unfolding rate in urea was only slightly affected by the additives, with the exception of GdmCl which enhanced the unfolding rate. The refolding rate was decreased in urea by EtOH and GdmCl, in contrast to glycerol and Na2SO4 which increased the refolding rate. At high temperature the unfolding was affected only slightly by the additives, except for GdmCl, and to a lesser extent NaCl, which enhanced the unfolding rate. The refolding rates were greatly decreased by Na2SO4, EtOH, and GdmCl, whereas glycerol and Nacl enhanced the process. It appears that 100 mM NaCl functions as a catalyst for the temperature-induced process, enhancing both the unfolding and refolding rates. The results indicate that the mechanisms of CIP unfolding and refolding are similar in urea and at high temperature but different at high pH.
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PMID:Unfolding and refolding of Coprinus cinereus peroxidase at high pH, in urea, and at high temperature. Effect of organic and ionic additives on these processes. 865 38

Urea-denatured alpha-zein was almost completely hydrolyzed into small peptides by digestion with 1/100 (w/w) of thermolysin at 37 degrees C for 3h. The angiotensin I-converting enzyme (ACE) inhibitory activity (IC50) of the thermolysin digest of total alpha-zein was 24.5 micrograms/ml, and most of the peptide fractions from Z19 alpha-zein and total alpha-zein separated by reverse-phase HPLC had more or less ACE inhibitory activity. From these fractions, thirty-six peptides, including 5 dipeptides, 14 tripeptides, 9 tetrapeptides, 5 pentapeptides, and 3 hexapeptides, were purified and their amino acid sequences were determined.
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PMID:Isolation from alpha-zein of thermolysin peptides with angiotensin I-converting enzyme inhibitory activity. 882 36

Pepsin successfully catalyzed the synthesis of several hydrophobic octa- and decapeptides in dimethylformamide-water solutions containing concentrated urea at pH 4.65. The factors that influence peptide synthesis in the presence of urea were studied using condensation of the tripeptides Z-Ala-Ala-Phe-OH and H-Leu-Ala-Ala-OCH3 as a model. The dependence of Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 yield on pepsin concentration and pH, as well as the behavior of pepsin during peptide synthesis were studied. It was shown that pepsin catalyzed the synthesis of Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 in guanidine hydrochloride and sodium dodecyl sulfate solutions. Other proteinases, subtilisin and thermolysin, were applied for the synthesis of p-nitroanilides of tri- and tetrapeptides in urea solutions. Proteinase-catalyzed peptide synthesis in the presence of denaturing agents might help to overcome the limitations caused by poor solubility of the starting peptide derivatives, although this effect is sometimes counterbalanced by the product solubility.
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PMID:Proteinase-catalyzed peptide synthesis in concentrated solutions of urea and other denaturing agents. 890 96

Reversible conversion between the native and scrambled proteins can be applied to analyze the denaturation curve of a disulfide-containing protein. In the case of RNase A, scrambled species could not be well separated from the native species by HPLC to permit precise quantitative analysis of the extent of denaturation. Methods are developed here to overcome this problem. The methods exploit the difference of conformational stability between the native and scrambled RNase A. When a sample of partially denatured RNase A was placed under mild reducing conditions (0.2-1 mM dithiothreitol for 10 min), the disulfide bonds of the native RNase A remain intact, whereas those of scrambled isomers become fully reduced. The native and fully reduced species of RNase A can be completely separated by HPLC. Alternatively, a mixture of partially denatured RNase A can be treated with mild concentration of proteolytic enzymes (trypsin or thermolysin). In this approach, scrambled isomers of RNase A were totally fragmented and readily separated from the native RNase A. These methods allow analysis and construction of the denaturation curves of RNase A in the presence of urea, GdmCl and GdmSCN.
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PMID:Quantitative analysis of the composition of the native and scrambled ribonuclease A. 1003 74

The method of limited proteolysis has proven to be appropriate for the determination of unfolding rate constants (k(U)) of ribonuclease A in the transition region of thermal denaturation [Arnold, U. & Ulbrich-Hofmann, R. (1997) Biochemistry 36, 2166-2172]. The aim of the present paper was to extend this procedure to the pretransition region of thermally and urea-induced denaturation where spectroscopic methods do not allow direct measurement of k(U). The results show that the approach can be applied successfully to denaturing (free energy of unfolding Delta G < 10 kJ.mol(-1)) and to marginally native conditions (Delta G = 10-25 kJ.mol(-1)). Under moderately (Delta G = 25-30 kJ.mol(-1)) and strongly native conditions (Delta G > 30 kJ.mol(-1)), however, the determination of kU was not possible in this way as the proteolytic degradation of ribonuclease A by thermolysin or trypsin was no longer determined by global unfolding. Here, proteolysis proceeds via the native RNase A. In the presence of low concentrations of urea, the rate constants of proteolysis were, surprisingly, smaller than in the absence of urea. As the protease activity has been taken into account, this result points to a local stabilization of the RNase A molecule.
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PMID:Proteolytic degradation of ribonuclease A in the pretransition region of thermally and urea-induced unfolding. 1112 Nov 7

The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein.
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PMID:A tetrodotoxin-binding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties. 1217 12

The cyclotides constitute a recently discovered family of plant-derived peptides that have the unusual features of a head-to-tail cyclized backbone and a cystine knot core. These features are thought to contribute to their exceptional stability, as qualitatively observed during experiments aimed at sequencing and characterizing early members of the family. However, to date there has been no quantitative study of the thermal, chemical, or enzymatic stability of the cyclotides. In this study, we demonstrate the stability of the prototypic cyclotide kalata B1 to the chaotropic agents 6 M guanidine hydrochloride (GdHCl) and 8 M urea, to temperatures approaching boiling, to acid, and following incubation with a range of proteases, conditions under which most proteins readily unfold. NMR spectroscopy was used to demonstrate the thermal stability, while fluorescence and circular dichroism were used to monitor the chemical stability. Several variants of kalata B1 were also examined, including kalata B2, which has five amino acid substitutions from B1, two acyclic permutants in which the backbone was broken but the cystine knot was retained, and a two-disulfide bond mutant. Together, these allowed determinations of the relative roles of the cystine knot and the circular backbone on the stability of the cyclotides. Addition of a denaturant to kalata B1 or an acyclic permutant did not cause unfolding, but the two-disulfide derivative was less stable, despite having a similar three-dimensional structure. It appears that the cystine knot is more important than the circular backbone in the chemical stability of the cyclotides. Furthermore, the cystine knot of the cyclotides is more stable than those in similar-sized molecules, judging by a comparison with the conotoxin PVIIA. There was no evidence for enzymatic digestion of native kalata B1 as monitored by LC-MS, but the reduced form was susceptible to proteolysis by trypsin, endoproteinase Glu-C, and thermolysin. Fluorescence spectra of kalata B1 in the presence of dithiothreitol, a reducing agent, showed a marked increase in intensity thought to be due to removal of the quenching effect on the Trp residue by the neighboring Cys5-Cys17 disulfide bond. In general, the reduced peptides were significantly more susceptible to chemical or enzymatic breakdown than the oxidized species.
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PMID:Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. 1514 80

Glucose-6-phosphate dehydrogenase (G6PDH) from hepatopancreas of the land snail, Otala lactea, shows distinct changes in properties between active and estivating (dormant) states, providing the first evidence of pentose phosphate cycle regulation during hypometabolism. Compared with active snails, G6PDH Vmax increased by 50%, Km for glucose-6-phosphate decreased by 50%, Ka Mg x citrate decreased by 35%, and activation energy (from Arrhenius plots) decreased by 35% during estivation. DEAE-Sephadex chromatography separated two peaks of activity and in vitro incubations stimulating protein kinases or phosphatases showed that peak I (low phosphate) G6PDH was higher in active snails (57% of activity) whereas peak II (high phosphate) G6PDH dominated during estivation (71% of total). Kinetic properties of peaks I and II forms mirrored the enzyme from active and estivated states, respectively. Peak II G6PDH also showed reduced sensitivity to urea inhibition of activity and greater stability to thermolysin protease treatment. The interconversion of G6PDH between active and estivating forms was linked to protein kinase G and protein phosphatase 1. Estivation-induced phosphorylation of G6PDH may enhance relative carbon flow through the pentose phosphate cycle, compared with glycolysis, to help maintain NADPH production for use in antioxidant defense.
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PMID:Glucose-6-phosphate dehydrogenase regulation during hypometabolism. 1625 36


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