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)

1. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) at a concentration of 0.5 mM had no effect on the serine proteinases plasma kallikrein and leucocyte elastase or the metalloproteinases thermolysin and clostridial collagenase. In contrast, 10 muM-E-64 rapidly inactivated the cysteine proteinases cathepsins B, H and L and papain (t0.5 = 0.1-17.3s). The streptococcal cysteine proteinase reacted much more slowly, and there was no irreversible inactivation of clostripain. The cysteine-dependent exopeptidase dipeptidyl peptidase I was very slowly inactivated by E-64. 2. the active-site-directed nature of the interaction of cathepsin B and papain with E-64 was established by protection of the enzyme in the presence of the reversible competitive inhibitor leupeptin and by the stereospecificity for inhibition by the L as opposed to the D compound. 3. It was shown that the rapid stoichiometric reaction of the cysteine proteinases related to papain can be used to determine the operational molarity of solutions of the enzymes and thus to calibrate rate assays. 4. The apparent second-order rate constants for the inactivation of human cathepsins B and H and rat cathepsin L by a series of structural analogues of E-64 are reported, and compared with those for some other active-site-directed inhibitors of cysteine proteinases. 5. L-trans-Epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475) was found to inhibit cathepsins B and L more rapidly than E-64. 6. Fumaryl-leucylamido(3-methyl)butane (Dc-11) was 100-fold less reactive than the corresponding epoxide, but was nevertheless about as effective as iodoacetate.
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PMID:L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. 704 72

Two approaches were used to establish the intercellular distribution of fatty acid synthetase and thioesterase II in the lactating rat mammary gland. Thioesterase II is the chain-length regulatory enzyme in the biosynthesis of the medium-chain fatty acids characteristic of milk fat. Using immunohistochemical techniques, immunoreactive fatty acid synthetase was found in both mammary adipocytes and epithelial cells; in contrast, immunoreactive epithelial cells were isolated from lactating rat mammary glands after digestion with collagenase and thermolysin, and their lipogenic activity was studied using isotopically labelled acetate. Consistent with the immunohistochemical data, adipocytes synthesized exclusively long-chain fatty acids whereas epithelial cells synthesized predominantly chain fatty acids. The results indicate that the capacity for synthesis of medium-chain fatty acids is a unique property of the epithelial cell component of the mammary gland.
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PMID:Localization of thioesterase II, the chain-length regulatory enzyme of milk fatty acid synthesis, in rat mammary gland epithelial cells. 705 Feb 81

In rheumatoid and osteoarthritis, degradation of articular cartilage is mediated by the matrix metalloproteinases collagenase, stromelysin and gelatinase. The key event in this process is the cleavage of triple helical collagen by collagenase. We have determined the crystal structure of the catalytic domain of human recombinant fibroblast collagenase complexed with a synthetic inhibitor at 2.2 A resolution. The protein fold is similar to the amino termini of the zinc endopeptidases astacin thermolysin and elastase despite a lack of primary sequence homology. The conformation of the bound inhibitor provides a molecular basis for the design of inhibitors of collagenase and other matrix metalloproteinases. Such inhibitors should be useful in the treatment of a variety of diseases including arthritis and cancer.
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PMID:Structure of the catalytic domain of human fibroblast collagenase complexed with an inhibitor. 765 13

Collagenase is a member of the matrix metalloproteinase (MMP) family of enzymes. Aberrant regulation of this family has been implicated in pathologies such as arthritis and metastasis. Two crystal forms of the catalytic (19-kDa) domain of human fibroblast collagenase have been determined using collagenase complexed with a peptide-based inhibitor (CPLX) as a starting model [Lovejoy et al. (1994) Science 263, 375]. The first crystal form (CF1) contains one molecule in the asymmetric unit and has been determined at 1.9-A resolution with an R factor of 19.8%. The second crystal form (CF2) contains two molecules (A and B) in the asymmetric unit and has been determined at 2.1-A resolution with an R factor of 19.7%. The catalytic domain of collagenase is spherical with an active site cleft that contains a ligated catalytic zinc ion. Collagenase shares some structural homology with the bacterial zinc proteinase, thermolysin [Matthews et al. (1972) Nature, New Biol. 238, 37], and the crayfish digestive peptidase, astacin [Bode et al. (1992) Nature 358, 164]. The amino terminus (Leu 102 to Gly 105) of CF1 and CF2 molecules A and B differs from the conformation found in CPLX by bending away from the molecule and interacting with the active site cleft of symmetry-related molecules. In this alternative conformation, both the mainchain nitrogen and carbonyl oxygen of Leu 102 ligate the symmetry-related catalytic zinc. Although there are structural differences in the active site clefts of CF1, CF2, and CPLX, a number of complex-stabilizing interactions are conserved. The structure of collagenase will be useful for developing compounds that selectively inhibit individual members of the closely related matrix metalloproteinase family.
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PMID:Crystal structures of recombinant 19-kDa human fibroblast collagenase complexed to itself. 803 54

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 A resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded beta-sheet and three alpha-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase.
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PMID:1.56 A structure of mature truncated human fibroblast collagenase. 809 Jul 13

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

Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.
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PMID:Structure of the catalytic domain of fibroblast collagenase complexed with an inhibitor. 827 10

Substrate specificity studies from this laboratory suggested that Ac-Pro-Leu-Ala-Nva-TrpNH2, and its thioester derivative, Ac-Pro-Leu-Ala-SNva-TrpNH2, would be substrates for stromelysin (SLN). In this paper, we report that both peptides are efficiently hydrolyzed not only by SLN but also by two other matrix metalloproteinases, collagenase and gelatinase, and by the bacterial metalloproteinase thermolysin. The pH-dependence of kc/Km for the SLN-catalyzed hydrolysis of Ac-Pro-Leu-Ala-SNva-TrpNH2 is identical to pH-dependencies for peptide hydrolysis and suggests no major mechanistic differences between thioester and amide hydrolysis by SLN.
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PMID:Thioester hydrolysis by matrix metalloproteinases. 831 64

Inhibitory effects of some enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting were investigated. The hydrolysate of porcine skin collagen with thermolysin or bacterial collagenase inhibited fibrinogen/thrombin clotting, but did not inhibit the activity of thrombin. Although the activity was not pronounced, the hydrolysate of collagen with such proteinases as trypsin and pepsin also inhibited the clotting. Gly-Pro-Arg, which is a known inhibitor of fibrinogen/thrombin clotting, was isolated from the bacterial collagenase hydrolysate of porcine collagen by HPLC. Collagen-related synthetic peptides such as Gly-Pro-Arg-Gly, Gly-Pro-Arg-Gly-Pro, Gly-Pro-Arg-Gly-Pro-Ala, Gly-Pro-Arg-Gly-Pro-Pro, and Gly-Pro-Arg-Pro-Pro also inhibited the clotting, but did not inhibit the activity of thrombin. The inhibitory activity of Gly-Pro-Arg-Gly-Pro-Pro and Gly-Pro-Arg-Pro-Pro was more marked than that of Gly-Pro-Arg. However, Gly-Pro-Lys, Gly-Ala-Arg, Gly-Pro-Hyp, Ala-Gly-Pro-Arg and Gly-Pro-Ala-Gly-Pro-Arg had no inhibitory effect on the clotting.
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PMID:Inhibitory effects of enzymatic hydrolysates of collagen and collagen-related synthetic peptides on fibrinogen/thrombin clotting. 832 52

The proteolytic enzyme stromelysin-1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin-1 is synthesized as a pro-enzyme that is activated by removal of an N-terminal prodomain. The active enzyme contains a catalytic domain and a C-terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three-dimensional structures of both a C-truncated form of the proenzyme and an inhibited complex of the catalytic domain by X-ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three alpha-helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino-to-carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.
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PMID:Stromelysin-1: three-dimensional structure of the inhibited catalytic domain and of the C-truncated proenzyme. 853 33


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