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)

A metalloprotease was isolated from the culture medium of a mutant of Staphylococcus aureus strain V8. The enzyme had a molecular weight of 38,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an optimum pH of 7.0 and exhibited a specificity for peptide bonds on the N-terminal side of large hydrophobic residues. The protease was fully inactivated by 0-phenanthroline but could be reactivated by zinc ions. Cobalt may be substituted for zinc, producing an activity which corresponds to 160% of that of the native enzyme. All these data indicate that this protease is a typical bacterial neutral metalloprotease. The role of this metalloprotease in the activation of the precursor of another protease secreted by the same organism, staphylococcal protease, has been identified. Mutants which lack the metalloprotease accumulated the precursor, which can be specifically activated by the addition of the purified metalloprotease or the related enzyme thermolysin. The purification of the precursor is also reported.
 ...
PMID:Role of metalloprotease in activation of the precursor of staphylococcal protease. 71 76

The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var. amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.
 ...
PMID:Degradation of streptomyces metalloprotease inhibitor (SMPI) by neutral protease from Bacillus subtilis var. amylosacchariticus. 136 40

To further analyze CD10/NEP function in lymphoid and nonlymphoid cells using well characterized murine systems, we isolated the murine CD10/NEP homologue, determined its chromosomal location, and modeled the enzyme's active site. The murine CD10/NEP cDNA predicts a 750-amino acid (aa) type II integral membrane protein with 90% identity to the human CD10 sequence and 100% conservation of critical aa and functional motifs. The latter include the pentapeptide consensus sequence required for zinc binding and catalytic activity, additional aa associated with substrate binding, and the extracellular cysteines that participate in disulfide bonds required for enzymatic activity. Like its human homologue, murine CD10/NEP has multiple alternative 5'-untranslated region sequences. The gene is localized on the proximal half of murine chromosome 3. In Northern analysis, murine CD10/NEP transcripts are abundant in bone marrow stromal cells that support pre-B cell differentiation but are undetectable in representative Abelson transformed pre-B cell lines. The murine CD10/NEP active site was modeled by aligning critical conserved CD10/NEP residues with comparable residues in the active site of thermolysin, a bacterial metalloprotease with similar substrate specificity. The model predicts that the two enzymes have similar clefts that comprise the active site and permit zinc-dependent substrate interactions.
 ...
PMID:Murine common acute lymphoblastic leukemia antigen (CD10 neutral endopeptidase 24.11). Molecular characterization, chromosomal localization, and modeling of the active site. 137 1

Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an alpha-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an alpha-helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.
 ...
PMID:Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom. 143 99

A skimmed-milk clearing assay was used to identify, in a multicopy Streptomyces lividans 66 genomic library, DNA fragments that lead to increased expression of protease activity in S. lividans 66. Three independent loci were identified. The majority class (slpA, which represented 68 of 71 clones) produced large zones of clearing. Two other classes (designated slpB and slpC) showed smaller zones than slpA. Subcloning and deletion analysis of the slpA locus delineated the relevant DNA to within a 2.5 kilobase pair fragment. DNA sequence analysis revealed a structural gene associated with the appearance of an extracellular protein in the culture medium. The derived amino acid sequence indicated the presence of a zinc-binding motif, which was previously noted to be characteristic of metalloprotease enzymes. However, the relatively small size of the protein (apparent molecular weight 20,000-24,000) suggests that it represents a novel class of neutral proteases distinct from the thermolysin-type enzymes. An adjacent divergent open reading frame was identified and shown to cause a significant increase in protease activity when present together with the protease structural gene on a multicopy plasmid in S. lividans 66. The derived amino acid sequence of this open reading frame showed homology with previously characterized regulatory proteins of the LysR family of transcriptional regulator proteins.
 ...
PMID:Cloning of genetic loci involved in endoprotease activity in Streptomyces lividans 66: a novel neutral protease gene with an adjacent divergent putative regulatory gene. 146 66

The elastase protein of Pseudomonas aeruginosa is a zinc metalloprotease which has been shown to be a member of the bacterial neutral protease family. Its overall tertiary structure is similar to that of thermolysin. The x-ray crystallographic structure of the elastase has been solved to high resolution in three different crystal forms. Substantial conformational differences are observed in the protein in different crystal forms. In the absence of ligand, and independently in the presence of a covalent noncompetitive inhibitor, the elastase is observed to have a relatively "open" substrate binding cleft, while in the presence of tight-binding competitive inhibitors, the active site cleft is "closed".
 ...
PMID:Crystallographic structures of the elastase of Pseudomonas aeruginosa. 148 11

A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85 degrees C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40 degrees C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.
 ...
PMID:Purification and characterization of a thermostable carboxypeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. 159 79

Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.
 ...
PMID:Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11. 167 40

By a computer analysis of the five Bacilli metalloprotease sequences it was found that mesophilic Bacillus amyloliquefaciens and B. subtilis proteases had lost two Ca(2+)-binding sites due to the substitutions Asp----Ser 57, Asp----Thr 59, Asp----Pro 200, in comparison with the thermostable B. thermoproteolyticus thermolysin and B. stearothermophilus protease, which conserved three Ca(2+)-binding sites, and B. cereus protease with the intermediate thermostability, which had presumably lost only one site (Ile----Lys 197 substitution). The multiple substitutions within the sequence regions 91-101, 150-154, and 275-280 of the mesophilic enzymes also corresponded with the decrease in the proteinase thermostability value. On the known X-ray structure of thermolysin these sequence regions are spatially drawn together, being located near the central alpha-helix opposite the active site hole and providing the contact of the N- and C-terminal domains. It may be concluded that to increase the thermostability of the mesophilic Bacilli proteinases it is necessary to substitute the sequence regions 91-101, 150-154, 275-280 for the thermolysin ones and restore the Ca(2+)-binding sites of the enzymes.
 ...
PMID:Sequence regions of Bacilli metalloproteinases that can affect enzyme thermostability. 181 91

The entire nucleotide sequence of an open reading frame located immediately downstream of the listeriolysin gene from a virulent Listeria monocytogenes serotype 1/2a strain was determined. The product of the open reading frame was 510 amino acids with a predicted molecular weight of 57,400. The deduced amino acid sequence of this open reading frame is highly similar to that of a family of secreted metalloproteases produced by various members of the genus Bacillus, of which thermolysin is the prototype. Immunoblots performed with specific antisera raised against thermolysin from Bacillus stearothermophilus allowed the detection of a 60-kDa polypeptide, corresponding to the pro-form of the protease, in culture supernatants of L. monocytogenes strains. In maxicell experiments, Escherichia coli recombinants harboring this open reading frame also specifically directed production of a 60-kDa protein. Protease activity was low to undetectable in both Listeria strains and E. coli recombinants. This is due to lack of processing of the inactive pro-form of the protease to its mature active form in both species. We have designated this gene mpl for metalloprotease of L. monocytogenes. The gene was present only in pathogenic L. monocytogenes strains, in which it was physically linked to the listeriolysin gene.
 ...
PMID:Molecular cloning, sequencing, and identification of a metalloprotease gene from Listeria monocytogenes that is species specific and physically linked to the listeriolysin gene. 189 3


1 2 3 4 5 6 7 8 9 10 Next >>