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

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. 156 52

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

Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release. To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli. After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing. It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-residue synthetic polypeptide spanning the cleavage site of the substrate. The importance of Glu234 in the catalytic activity of the light chain, possibly analogous to Glu143 of thermolysin, was examined using site-directed mutagenesis. Changing Glu234 to Ala abolished the protease activity of the light chain, but its ability to bind the polypeptide substrate was retained. Each recombinant light chain could be reconstituted with the heavy chain of tetanus toxin, yielding the same level of disulfide-linked species as the two native chains. Whereas the toxin formed with wild-type light chain exhibited appreciable neuromuscular paralysis activity and mouse lethality, the equivalent dichain material containing the Ala234 mutant lacked neurotoxicity in both the in vitro and in vivo assays. Thus, these results demonstrate directly, for the first time, that the lethality of tetanus toxin and its inhibition of exocytosis in intact neurons are attributable largely, if not exclusively, to endoprotease activity.
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PMID:A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstitution with native heavy chain. 791 29

Flavobacterium meningosepticum, Elder strain (ATCC 33958), secretes into the medium a neutral zinc endoprotease as a major component of the extracellular proteins. The enzyme was purified to homogeneity in a simple two-step procedure involving ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular weight of this metalloprotease was determined to be about 27,000 (P27) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P27 was comparable to thermolysin in the relative rates of elastin-orcein, azocasein, and azoalbumin hydrolysis. P27 and thermolysin hydrolyzed equally well 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the same primary sites that are susceptible to cleavage by vertebrate collagenases, Gly-Ile, and Gly-Leu. P27 was also capable of partially hydrolyzing Type I acid-soluble calf skin collagen and slowly hydrolyzing N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala, a bacterial collagenase substrate not cleaved by thermolysin. P27 was further differentiated from thermolysin from the inability of the former to hydrolyze N-[3-(2-furyl)acryloyl]-Gly-Leu-NH2. In addition, a vertebrate elastase substrate succinyl-Ala-Ala-Ala-p-nitroanilide was hydrolyzed by P27 but not by thermolysin. P27 is a newly described and unique enzyme from the standpoint of substrate specificity and from the fact that it is resistant to inhibition by phosphoramidon, an inhibitor of a number of zinc endopeptidases, including thermolysin.
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PMID:Purification and characterization of a neutral zinc endopeptidase secreted by Flavobacterium meningosepticum. 818 8

Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane-bound Zn-metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc-endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site-directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP-Gln711 and NEP-Phe711). Determination of kinetic parameters showed that both mutants had Km values very similar to that of the non-mutated enzyme but that their kcat values were 25-fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.
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PMID:Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state. 844 Mar 86

The role of the C-terminal domain of rhodopsin in the activation of transducin was studied. The treatment of photoreceptor membranes with trypsin, thermolysin, and Asp-N-endoprotease led to the respective rhodopsin species devoid of 9, 12-, or 19-aa C-terminal fragments. It was shown that the removal of 9-aa fragment by trypsin does not affect the catalytic activity of the receptor, whereas the thermolysin-induced truncation of the rhodopsin C-terminus by 12 aa about 1.5-fold enhances its activity. The Asp-N-endoprotease-assisted removal of 19 aa (i.e., the shortening by seven more C-terminal aa) virtually unchanges the rhodopsin catalytic activity compared to the preparation truncated with thermolysin. These results suggest that the part of the rhodopsin C-terminal fragment between the sites of its cleavage by trypsin and thermolysin (Val337-Ser338-Lys339) inhibits the signal transduction from rhodopsin to the next component of visual cascade. The English version of the paper.
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PMID:[The effect of proteolytic removal of the C-terminal fragment of rhodopsin on its ability to activate visual cascade]. 1187 69

A new fluorogenic, lanthanide-based oligopeptide substrate for the detection of the zinc-dependent endoprotease thermolysin is described. Using time-resolved fluorescence measurement, a highly sensitive assay for thermolysin was developed with a 50pM detection limit (3.5fmol).
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PMID:Lanthanide-based fluorogenic peptide substrate for the highly sensitive detection of thermolysin. 1972 May 29