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)

The cloned npr gene of Streptomyces cacaoi encodes a 60-kDa protein (prepro-Npr) consisting of a typical secretory signal peptide, a propeptide (22 kDa), and the 35-kDa mature metalloprotease (Npr). The maturation of Npr occurs extracellularly via an autocatalytic cleavage of the secreted intermediate pro-Npr (Chang, P.C., and Lee, Y.-H.W. (1992) J. Biol. Chem. 267, 3952-3958). In this study, we investigated the roles of the propeptide in the maturation and secretion of Npr. Partial deletion of the propeptide region while leaving the signal peptide and the mature Npr sequence intact all led to abolishment of Npr activity and caused concomitant slight and transient accumulation of low molecular weight forms of Npr or pro-Npr derivatives extracellularly. The intact propeptide and its truncated form alone could be secreted into the medium if their NH2 termini were directly fused with the signal peptide sequence of Npr. However, similar fusion of the mature protease domain to the signal peptide without the propeptide sequence completely abolished the Npr production intracellularly and extracellularly. All these results demonstrate that the propeptide plays an important role in maturation and secretion of Npr protease, as in the case of alpha-lytic protease and subtilisin. In addition, our data suggest that an intact propeptide region is essential for the formation of mature active Npr, but not for the secretion of Npr and its derivatives. This distinguishes the maturation and secretion of S. cacaoi Npr from those of other propeptide-containing bacterial serine proteases and thermolysin-like protease.
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PMID:The roles of propeptide in maturation and secretion of Npr protease from Streptomyces. 810 97

Synthetic inhibitors of interstitial collagenase, tri- and tetrapeptidyl hydroxamic acids, have been developed and tested for their inhibitory activities against human matrix metalloproteinases. A water soluble inhibitor, p-NH2-Bz-Gly-Pro-D-Leu-D-Ala-NHOH (FN-439) inhibited interstitial and granulocyte collagenases, granulocyte gelatinase and skin fibroblast stromelysin with IC50 of 1 x 10(-6) M, 3.0 x 10(-5) M and 1.5 x 10(-4), respectively, but not thermolysin and serine proteinases. FN-439 was found to retain its inhibitory activity against matrix metalloproteinases even after prolonged incubation with pronase or human granulocyte elastase, indicating a favorite candidate of the inhibitor to modulate metalloproteinase activities in vivo.
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PMID:Inhibition of matrix metalloproteinases by peptidyl hydroxamic acids. 814 88

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

The sites and the sequence of the proteolytic cleavages of bovine, human and rat thyroglobulin, during the limited proteolysis with thermolysin and trypsin, were determined by sequencing the NH2 termini of the peptides produced and comparing them to the cDNA-derived sequences of bovine, human and rat thyroglobulin. Major cleavage sites of bovine thyroglobulin included residues 240, 502, 993, 1218, 1784 with thermolysin, and 240, 520, 1142, 1783, 2515 with trypsin. Cleavage sites of human thyroglobulin included residues 503, 982, 990, 1405, 1831 with thermolysin, and 522, 1627, 2513 with trypsin. Those of rat thyroglobulin included residues 501, 1776, 1784 with thermolysin, and 522, 1771, 1825, 2515 with trypsin (numbered as in bovine thyroglobulin). Thus, thyroglobulin from various species presents well localized and conserved regions particularly sensitive to proteolysis. The most sensitive region extended for 30 residues after residue 500. Another major cluster of cleavages was centered around residue 1800; this region was only partially sensitive in human thyroglobulin. A conserved tryptic site lay at the COOH terminus of the molecule. Most cleavage sites occurred within the inserted sequences that disrupt the Cys-rich, tandem repeats of thyroglobulin and either contain or are located near exon-intron junctions. Several cleavage sites lay in proximity of early iodinated or hormonogenic tyrosyl residues or of putative N-linked glycosylation sites. While a predominantly beta-type secondary structure and a rigid three-dimensional structure were predicted for the Cys-rich repeats, stretches of predicted alpha-helices, beta-strands and irregular structure were interspersed in the regions surrounding the cleavage sites. These data demonstrate the existence of conserved regions of thyroglobulin inherently sensitive to proteolysis, which most likely represent solvent-exposed regions of the primary structure, possibly forming loops at the surface of thyroglobulin.
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PMID:Preferential sites of proteolytic cleavage of bovine, human and rat thyroglobulin. The use of limited proteolysis to detect solvent-exposed regions of the primary structure. 826 51

The receptor for human urokinase-type plasminogen activator (uPAR) is synthesized as a 313-residue-long polypeptide containing 28 cysteine residues, the pattern of which defines three homologous repeats within the protein. These entities are believed to represent a novel protein domain structure, of which the NH2-terminal domain of uPAR can be covalently cross-linked to the epidermal growth factor-like module of urokinase after receptor-ligand interaction. The NH2-terminal domain of a recombinant, soluble uPAR derivative, labeled with [35S]cysteine, was isolated after limited proteolysis with chymotrypsin. The four disulfide bonds present within this domain were assigned by a combination of plasma desorption mass spectrometry, amino acid composition, and sequence analyses of peptides generated by trypsin, endoproteinase Asp-N, and thermolysin. The following disulfide bond structure was determined: Cys3-Cys24, Cys6-Cys12, Cys17-Cys45, and Cys71-Cys76. Similar cysteine pairing is likely to be found within other members of this protein superfamily, i.e. the membrane inhibitor of reactive lysis, Ly-6, and the remaining two domains of uPAR. However, an additional pair of cysteines present within these domains probably forms a fifth disulfide bond.
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PMID:Localization of the disulfide bonds in the NH2-terminal domain of the cellular receptor for human urokinase-type plasminogen activator. A domain structure belonging to a novel superfamily of glycolipid-anchored membrane proteins. 839 46

Recombinant human fibroblast pro-MMP-3 (prostromelysin-1) expressed in Chinese hamster ovary cells and the zymogen from cultured human dermal fibroblasts have been purified by monoclonal antibody immunoaffinity chromatography, and the role of Ca2+ in proenzyme activation and thermostability of the low mass catalytic domain of MMP-3 has been investigated. In the presence of high Ca2+ (5.0 mM), the organomercurial aminophenylmercuric acetate (APMA) initiated the stepwise removal of both NH2- and COOH-terminal domains from both recombinant and dermal fibroblast proenzymes, resulting in the generation of a heterogeneous family of nonglycosylated low mass truncated active enzyme species beginning at Phe83. However, in the presence of low Ca2+ (0.1 mM), incubation of recombinant pro-MMP-3 with or without APMA did not result in formation of either the high or low mass forms of active MMP-3 but resulted in complete autolysis of both enzyme species. The concentration of Ca2+ required for optimal pro-MMP-3 activation and stability of the low mass catalytic domain was 2.0 mM. The low mass truncated enzyme species containing the catalytic domain were remarkably heat-stable (90 min at 60 degrees C) in high Ca2+ (5.0 mM) but rapidly autolyzed when heated at 60 degrees C in low Ca2+ (0.1 mM). The thermostability properties of MMP-3 appeared to be specific for Ca2+, since no other divalent metal ions tested were able to confer thermostability to the low mass catalytic domain of MMP-3. From homology to the thermostable bacterial metalloprotease, thermolysin, two putative Ca2+ binding sites were found in the catalytic domain of MMP-3 and several other members of the MMP gene family. These putative Ca2+ binding sites are postulated to play an important role in stabilizing active MMP-3 and other members of the matrix metalloprotease gene family by protecting them against autolysis.
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PMID:Recombinant Chinese hamster ovary cell matrix metalloprotease-3 (MMP-3, stromelysin-1). Role of calcium in promatrix metalloprotease-3 (pro-MMP-3, prostromelysin-1) activation and thermostability of the low mass catalytic domain of MMP-3. 844 Jul 30

Inhibition of envelysin, a metalloproteinase which dissolves the fertilization envelope of sea urchin embryo, was studied using a synthetic autoinhibitor peptide. Ac-Pro-Arg-Cys-Gly-Val-Pro-Asp-Val-NH2, with a 'cysteine-switch' consensus sequence. Although its effect is reversible, the hatching of sea urchin embryos was effectively delayed by 0.5 mM of the peptide. When alpha 1-proteinase inhibitor was used as the substrate, envelysin was inhibited by the autoinhibitor and an Ala6 analogue, but not by a D-Cys3 analogue. However, envelysin was weakly inhibited by both D- and L-cysteines to the same extent. Snake venom alpha-protease exhibited cleavage and inhibition behavior similar to envelysin with a little weaker stereo-specificity. The results suggest that the coordination of the autoinhibitor Cys residue with the envelysin active site Zn is established only after the amino acid residues on both sides of the Cys residue get into an appropriate interaction with the catalytic site residues, and that the precise orientation of the cysteine SH group is essential. By contrast, thermolysin was weakly inhibited by the three peptide non-stereo-specifically. Furthermore, thermolysin cleaved the autoinhibitor at the Cys3 Gly4 bond when incubated without substrate.
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PMID:Stereo-specific inhibition of sea urchin envelysin (hatching enzyme) by a synthetic autoinhibitor peptide with a cysteine-switch consensus sequence. 846 15

The major surface glycoprotein of Leishmania promastigotes, referred to as GP63, is a zinc metalloproteinase of 63,000 M(r) containing a glycosylphosphatidylinositol (GPI) membrane anchor. Recent studies demonstrated that recombinant GP63 (rGP63) expressed by the baculovirus insect cell system was secreted as a glycosylated latent proteinase that required activation for full proteinase activity (Button et al. (1993) Gene 134, 75-81). To extend these studies, the active site of L. major GP63 was characterized by site-directed mutagenesis and the activation mechanism of latent rGP63 was studied using both secreted and cell surface expression systems. To determine whether the proposed active site of L. major GP63 conforms to other well characterized zinc metalloproteinases, the proposed GP63 catalytic Glu-265, corresponding to catalytic Glu-147 of thermolysin, was changed to Asp-265. Using a transient expression system in COS-7 cells, expression of the Asp-265 mutant GP63 gene resulted in rGP63 with no detectable proteinase activity, whereas expression of the wild-type GP63 gene resulted in rGP63 with a level of proteinase activity similar to native GP63. Thus, the mechanism of GP63 proteinase activity is predicted to be homologous to that of other well characterized zinc metalloproteinases. NH2-Terminal sequence analysis revealed that activation with HgCl2 resulted in removal of the pro region, ultimately generating the mature NH2-terminus. This processing included the removal of a conserved Cys residue (Cys-48) and occurred by a cis mechanism, since the addition of previously activated rGP63 did not lead to an enhancement of latent rGP63 proteinase activation. The mechanism of activation of GP63 is consistent with the cysteine switch mechanism proposed for matrix metalloproteinases and thus has been conserved from protozoa to mammals.
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PMID:Analysis of the active site and activation mechanism of the Leishmania surface metalloproteinase GP63. 851 3

Pseudomonas aeruginosa is a zinc metalloprotease which may be involved in many infection processes, especially in the lung. In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities. Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2 and X-AlaR using either thermolysin or P. aeruginosa elastase itself. In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-AlaNH2 was found to be the best substrate, giving a catalytic ratio kcat/KM of 8600 mM-1.s-1. The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower. The sensitivity of the conductimetric method was also demonstrated with as little as 1 nM elastase (0.13 microgram), being easily and accurately detected (SD, 3.8% for 10 measurements). Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.
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PMID:Synthetic peptide substrates for a conductimetric assay of Pseudomonas aeruginosa elastase. 866 May 69

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 x 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu(Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenylhydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.
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PMID:Two amine oxidases from Aspergillus niger AKU 3302 contain topa quinone as the cofactor: unusual cofactor link to the glutamyl residue occurs only at one of the enzymes. 867 75


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