Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.23 (MMP)
 4,246 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.
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PMID:Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: importance of the N-terminal region between cysteine 3 and cysteine 13. 142 Jan 37

Human matrilysin devoid of its propeptide is expressed in Escherichia coli and purified to homogeneity by heparin chromatography after refolding of the guanidine hydrochloride solubilized protein. Matrilysin autolytically removes its N-terminal tripeptide Met-Tyr-Ser during the refolding process. The enzyme contains 1.91 +/- 0.08 zinc atoms/mol of protein and retains full activity when stored several months at 4 degrees C. It hydrolyzes the fluorescent substrate Dns-PLALWAR at the Ala-Leu bond with a kcat of 3.1 s-1 and K(m) of 1.8 x 10(-5) M at pH 7.5, 37 degrees C, values closely similar to those for the matrilysin produced by activation of the Chinese hamster ovary and E. coli-expressed promatrilysin. The properties of this form of matrilysin demonstrate that the propeptide is not essential for proper folding or stability of the enzyme but likely determines the N-terminal amino acid of the mature enzyme. The pH dependence of kcat/K(m) for Dns-PLALWAR shows that matrilysin has a broad pH optimum (5.0-9.0) and the pKa values obtained are 4.3 and 9.6 at 25 degrees C. The activity is inhibited by several metal binding agents including 1, 10-phenanthroline, OP, but not by the nonchelating isomer, 1,7-phenanthroline. OP inhibits instantaneously by likely forming a transient ternary enzyme.metal.chelator complex. The zinc atom is then removed from the protein in a time-dependent manner. In agreement with the kinetic studies, dialysis in the presence of OP and CaCl2 removes only the catalytic zinc atom. The monozinc enzyme can be reactivated to 90%, 56%, 27%, and 17% of the native activity by addition of zinc, manganese, nickel, and cobalt, respectively. Cadmium, on the other hand, forms an inactive Cd/Zn hybrid. The differences in the chelator accessibility properties of the two zinc sites can thus be exploited to yield metallohybrids of matrilysin.
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PMID:Metal and pH dependence of heptapeptide catalysis by human matrilysin. 896 47

The recombinant prepro-form of human matrix metalloproteinase 7 (matrilysin or MMP-7) was overexpressed in Escherichia coli as insoluble inclusion bodies. The recombinant protein was refolded by 100-fold dilution after solubilization with 6 M guanidine HCl. The refolding was monitored by the recovery of matrilysin activity. The addition of either 1.0 M arginine or 0.1% Brij-35 promoted remarkably the refolding. The refolding was dependent on pH and temperature, with lower temperature (<10 degrees C) and pH 6-8 preferable. Glutathione had no effect on refolding, and it was excluded from the refolding conditions. Starting with inclusion bodies (2.0 g, wet) containing 360 mg protein, 29.5 mg of pro-matrilysin (30 kDa) was obtained after refolding with 1.0% Brij-35 at pH 7.5 and 4 degrees C for 12 h. Pro-matrilysin (24.0 mg) was purified to homogeneity by cation-exchange HPLC with a 15-fold increase in purity and an activity yield of 81.3%. Pro-matrilysin was converted entirely to matrilysin (19.0 kDa; 15.2 mg) by activation with a mercuric reagent. The activity (k(cat)/K(m)) of matrilysin was 1.7 x 10(5) M(-1) x s(-1).
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PMID:Refolding and recovery of recombinant human matrix metalloproteinase 7 (matrilysin) from inclusion bodies expressed by Escherichia coli. 1054 84

States of tryptophyl residues and stability of human matrilysin were studied. The activation energy for the thermal inactivation of matrilysin was determined to be 237 kJ/mol, and 50% of the activity was lost upon incubation at 69 degrees C for 10 min. The activity was increased by adding NaCl, and was doubled with 3 M NaCl. Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues. Half of the change was observed at 2.2-2.7 M GdnHCl, whereas no change was observed even with 8 M urea. Half of the inactivation was induced at 0.8 M GndHCl and at 2 M urea. The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denaturation. Matrilysin contains four tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide. No tryptophyls in the native enzyme were accessible to I(-) and Cs(+), and 2.4 residues were accessible to acrylamide. Based on the crystallographic study, Trp154 is water-accessible, but it should be in a crevice not to contact with I(-) and Cs(+). All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one.
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PMID:States of tryptophyl residues and stability of recombinant human matrix metalloproteinase 7 (matrilysin) as examined by fluorescence. 1096 33