Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Genomic clones containing the complete gene encoding human fibroblast interstitial collagenase were isolated from a lambda phage human DNA library. The gene is comprised from 10 exons and spans 8.2 kilobase pairs. We have mapped the relative positions and determined the DNA sequence of all the exon/intron borders of the gene. The organization of the human interstitial collagenase gene is very similar to that of rabbit collagenase and of two other extracellular matrix (ECM) metalloproteases: rat stromelysin (transin) and rat transin 2. All four genes are organized into 10 exons of virtually identical size while the length of the 3' proximal introns is subject to variation. The protein sequence comprising the putative active center is coded for by exon 5 of all four genes and contains a strongly conserved zinc binding site. This observation suggests that the organization of the ECM metalloprotease genes reflect the structure of the functional domains of the enzyme proteins. The structural data accumulated so far provides evidence for the existence of a gene family coding for secreted ECM metalloproteases and suggests that gene duplication played an important role in its formation.
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PMID:The structure of the human skin fibroblast collagenase gene. 283 3

We have developed a system for studying the proteinase activity of a collagenase family member, transin. Cos cells transfected with a vector designed to direct synthesis of a secretable fusion protein between staphylococcal protein A and transin secrete a latent proteinase, activable by 4-aminophenylmercuric acetate, which binds to IgG-Sepharose. Treatment with 4-aminophenylmercuric acetate leads to cleavage of the fusion protein and elution of the active proteinase transin. Based on results obtained with this system we propose that transin comprises an N-terminal proteinase domain and an independent C-terminal hemopexin-like domain. The latter domain is not required for binding of inhibitors or for maintenance of transin in its inactive form. The sequence PRCGVPDV is present in the proenzyme forms of collagenase family proteinases just upstream from the N termini of the active enzymes. We show that mutations within this sequence lead to transin variants with a much increased tendency to undergo spontaneous activation. Finally, we show that mutations within a region of transin having sequence similarity to the zinc-binding site of bacterial metalloproteinases inactivate the proteinase activity of transin, lending support to the notion that this region represents part of transin's active site.
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PMID:Structure-function relationships in the collagenase family member transin. 284 36

Active site metal substitutions for both gamma- and zeta-collagenases from Clostridium histolyticum have been made by direct metal exchange. The incubation of Co(II), Cu(II), Ni(II), Cd(II), and Hg(II) with these native collagenases results in changes in activity that parallel those observed for the reconstitution of the respective apoenzymes with these metal ions. For both collagenases, the exchange reactions with Co(II) and Cu(II) are complete within 1 min. However, the changes in activity observed on addition of Ni(II), Cd(II), and Hg(II) to gamma-collagenase and Cd(II) and Hg(II) to zeta-collagenase are time dependent. The kinetic parameters Kcat and KM have been determined for each of the active metallospecies. The substitution of the active-site metal ion in gamma-collagenase results in changes in both kcat and KM, while the effect observed in zeta-collagenase is primarily on KM. This suggests that there are differences in the mechanisms of these two collagenases, at least with respect to the role of the zinc ion in catalysis.
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PMID:Preparation by direct metal exchange and kinetic study of active site metal substituted class I and class II Clostridium histolyticum collagenases. 284 92

A new series of thio ester, depsipeptide, and peptide substrates have been synthesized for the bacterial enzyme Clostridium histolyticum collagenase. The hydrolysis of the depsipeptide substrate was followed on a pH stat, and thio ester hydrolysis was measured by inclusion of the chromogenic thiol reagent 4,4'-dithiopyridine in the assay mixture. The best thio ester substrate, Boc-Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba, had a kcat/KM of 63 000 M-1 s-1, while several shorter thio ester sequences were inactive as substrates. In general, the peptide analogues of all the reactive thio ester substrates were shown to be hydrolyzed 5-10 times faster by collagenase. In one case (Z-Gly-Pro-Leu-Gly-Pro-NH2) where a comparison was made, the peptide substrate was respectively 8- and 106-fold more readily hydrolyzed than the corresponding thio ester and ester substrates. Cleavages of the two fluorescence-quench substrates Abz-Gly-Pro-Leu-Gly-Pro-Nba and Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba could be easily followed fluorogenically since a 5-10-fold increase in fluorescence occurred upon hydrolysis. The fluorescent peptide substrate is the best synthetic substrate known for C. histolyticum collagenase with a kcat/KM value of 490 000 M-1 s-1. A series of new reversible inhibitors were developed by the attachment of zinc ligating groups (hydroxamic acid, carboxymethyl, and thiol) to various peptide sequences specific for C. histolyticum collagenase. The shorter peptides designed to bind to either the P3-P1 or P1'-P3' subsites were poor to moderate inhibitors. The thiol HSCH2CH2CO-Pro-Nba had the lowest K1 (0.02 mM).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Clostridium histolyticum collagenase: development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors. 299 78

The collagenase from Clostridium histolyticum is a mixture of several collagenases, all of which are zinc metalloproteases. This enzyme catalyzes the cleavage of the X-Gly peptide bond in the repeating sequence of collagen: -Gly-Pro-X-Gly-Pro-X-. Thus the S3, S2, and S1 subsites on the enzyme appear to be occupied by the sequence -Gly-Pro-X- and the S1', S2', and S3' subsites also by -Gly-Pro-X-. Short peptides up to and including N alpha-acyltetrapeptides containing the repeat sequence do not detectably inhibit the enzyme (IC50 greater than 10 mM). However, peptide aldehydes of the form aminoacyl-X-glycinal, presumably occupying the S1, S2, ..., Sn subsites, are inhibitors. The most potent of these was Pro6-Gly-Pro-glycinal, with an IC50 of 340 +/- 70 microM. The single peptide aldehyde investigated, which could occupy the S1' and S2' subsites, 4-oxobutanoyl-L-proline, did not inhibit collagenase (IC50 greater than 20 mM). The peptide ketone 5-benzamido-4-oxo-6-phenylhexanoyl-Pro-Ala (XXV), which could occupy the S1-S3' subsites, inhibits collagenase with an IC50 of 120 +/- 50 microM, over 80-fold more potently than its parent peptide analogue benzoyl-Phe-Gly-Pro-Ala (XXIII). The alcohol analogue of XXV, 5-benzamido-4-hydroxy-6-phenylhexanoyl-Pro-Ala (XXVI), is over 60-fold less potent with an IC50 of 8 +/- 2mM. Extending the peptide ketone XXV to occupy the S2-S3' subsites gave 5-(N alpha-carbobenzoxy-L-prolinamido)-4-oxo-6-phenylhexanoyl-Pro -Ala (XXVII). Surprisingly, XXVII had an IC50 of only 5.2 +/- 2 mM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Aldehyde and ketone substrate analogues inhibit the collagenase of Clostridium histolyticum. 300 33

Co(II) ions increase the Vmax of clostridiopeptidase A, producing a maximum stimulation of overall enzymic activity of 120%. Co(II) does not displace Zn(II) from the active site, nor Ca(II) from its binding site on the enzyme. There appears to be an additional transition metal-binding site on clostridiopeptidase A, accepting Zn(II), which is inhibitory (Ki = 550 microM), or Co(II), which is stimulatory (Kact = 200 microM).
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PMID:Studies on the stimulation of the bacterial, collagenolytic enzyme clostridiopeptidase A by cobalt (II) ions. 300 84

A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.
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PMID:Functional constituents of the active site of human neutrophil collagenase. 301 Aug 66

A soil streptomycete designated as Streptomyces sp. A8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-a rginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration. The purified enzyme had an apparent molecular weight of about 75,000 by SDS-polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as alpha-alpha-dipyridyl and 8-hydroxyquinoline. Ethylene diamine tetraacetate completely inhibited the enzyme activity. Among the cations tested only Ca2+ and Mg2+ enhanced the collagenase activity. Heavy metal ions like Pb2+, Ag+, Cu2+ and Zn2+ strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca2+. Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.
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PMID:Purification and characterization of a streptomycete collagenase. 302 69

A comparison of the cDNA-derived amino acid sequences of human stromelysin and collagenase with the N-terminal sequences of purified enzymes reveals that these metalloproteinases are highly conserved and that they are secreted as proenzymes. A putative zinc-binding site was identified by its homology with the zinc-chelating sequence of thermolysin. These sequences permitted the identification of: transin, a protein induced in rat fibroblasts either exposed to growth factors or transformed by oncogenic viruses, as the rat homologue of stromelysin, and XHF1, a protein induced in human fibroblasts after treatment with tumourigenic agents, as collagenase.
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PMID:Comparison of human stromelysin and collagenase by cloning and sequence analysis. 303 Feb 90

Analysis of sequence alignments between the recently published sequence of human fibroblast collagenase and a computer file of published protease sequences has revealed a previously unrecognized homology to the 11 amino acids flanking the zinc-binding site of Serratia protease, a bacterial metalloprotease. There is also strong homology among several bacterial metalloproteases at this site. This finding implies that zinc binding by many proteins may have structural requirements which are apparent even in primary structure and which have been evolutionarily conserved or convergently evolved. This consensus sequence could be used as a marker for recognizing other members of the metalloprotease family.
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PMID:Human fibroblast collagenase contains an amino acid sequence homologous to the zinc-binding site of Serratia protease. 303 50


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