Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.11 (CD10)
9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on DNP-hexamethylenediamine-Sepharose and gramicidin S-Sepharose and gel filtration, a metalloproteinase was isolated from the cultural fluid of L. pneumophila (strain Philadelphia-1) grown for 20 hours. The enzyme was purified 1606-fold with a 31% yield. The enzyme has a Mr of 38,000, pI approximately 4.0 and optimum of proteolytic activity at pH 6.0-7.0, 55 degrees C. The proteinase is the most stable within the pH range of 6.0-9.0. The enzyme contains one atom of zinc per molecule. The amino acid composition of metalloproteinase is close to that of thermolysin and is characterized by a high methionine content--17 residues out of 348. In the B-chain of oxidized bovine insulin the enzyme hydrolyzes the bonds precedent to the amino groups of leucine, phenylalanine and tyrosine. The enzyme is inhibited by chelating agents--Na2-EDTA and o-phenanthroline as well as by diethylpyrocarbonate. The serine and thiol proteinase inhibitors do not influence the enzyme activity. Under the given conditions of cultivation metalloproteinase is the major endopeptidase produced by L. pneumophila. Thus, the proteolytic system of Legionelles is characterized by the combination of metalloproteinase and the earlier described phenylalanine aminopeptidase.
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PMID:[Extracellular metalloproteinase from Legionella pneumophila]. 366 68

This and two accompanying reports describe the intrinsic binding energy derived from a single hydrogen bond between an inhibitor and an enzyme. The results were obtained by comparing matched pairs of inhibitors of the zinc endopeptidase thermolysin that bind to the enzyme in an essentially identical manner but differ in the presence or absence of a specific hydrogen bond. This report describes five phosphorus-containing analogs of the peptides carbobenzoxy-Gly-Leu-X, in which the Gly-Leu peptide linkage is replaced with a phosphonate ester (-PO2(-)-O-). Values for the inhibition constants of these inhibitors show a direct relation with those of the corresponding phosphonamidate analogs (-PO2(-)-NH- in place of the Gly-Leu peptide moiety), which have been characterized previously as transition state analogs. However, each phosphonate ester is bound about 840 times more weakly than the analogous phosphonamidate, reflecting the loss of 4.0 +/- 0.1 kilocalories per mole in binding energy. From these results and the crystallographic analysis in the next report, it can be inferred that the value of 4.0 kilocalories per mole represents the intrinsic binding energy arising from a highly specific hydrogen binding interaction.
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PMID:Evaluation of intrinsic binding energy from a hydrogen bonding group in an enzyme inhibitor. 381 Jan 55

Neuronal and astroblast-rich cultures from rat brain degrade exogenously added substance P. The rate of degradation is decreased by diisopropylfluorophosphate, phosphoramidon and bacitracin, but not by N-ethylmaleimide or bestatin. When diisopropylfluorophosphate, phosphoramidon and bacitracin are simultaneously present in the culture medium, the degradation of substance P is completely inhibited. These results indicate that the hydrolysis of substance P by intact cells is catalyzed by the post-proline dipeptidylaminopeptidase (EC 3.4.14.5), the thermolysin-like metallopeptidase ("enkephalinase", EC 3.4.24.11) and a yet uncharacterized bacitracin-sensitive activity. While the thermolysin-like metallopeptidase is mainly associated with glial cells, the specific activity of the other enzymes is five times higher in the neuronal culture.
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PMID:Degradation of substance P by neurones and glial cells. 608 91

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.
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PMID:Degradation of neurotensin by rat brain synaptic membranes: involvement of a thermolysin-like metalloendopeptidase (enkephalinase), angiotensin-converting enzyme, and other unidentified peptidases. 630 59

Despite the similarities in their mechanism of action, the structural requirements for selective interaction with angiotensin-converting enzyme or enkephalinase are different. Inhibitory potency of a series of new mercaptoalkanoyl amino acids were determined on pure angiotensin-converting enzyme (EC 3.4.15.1) from porcine plasma and on neutral metalloendopeptidase (EC 3.4.24.11) purified from rat brain. This latter enzyme, first designated as enkephalinase, seems to be synaptically involved in the degradation of enkephalins. All tested compounds, whose design was based on the classical active-site model of metallopeptidases, are reversible and competitive inhibitors of both enzymes. Owing to the remarkable similarity in the general topology of metallopeptidases, the differences in optimal binding requirements to enkephalinase and angiotensin-converting enzyme were interpreted from crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. The large size of the S'1 subsite of enkephalinase allows efficient binding (Ki approximately equal to 2-30 nM) of aromatic and bulky hydrophobic residues such as a cyclohexyl ring. In contrast, a methyl group in position P'1 favors inhibitory potency against angiotensin-converting enzyme while a cyclohexyl ring leads to a complete loss of activity. This feature could mean that optimal binding of the Zn atom present in the catalytic site is a more stringent requirement in angiotensin-converting enzyme than in enkephalinase. An increase in the size of the P'2 component of thiol inhibitors potentiates the affinity for angiotensin-converting enzyme without a significant change on enkephalinase. Finally, methylation of the ultimate amide bond of inhibitors produces a 30-fold decrease in potency towards enkephalinase but does not affect the binding of angiotensin-converting enzyme. These findings allow a rational design of selective inhibitors of enkephalinase, an essential prerequisite for their possible clinical use as new analgesic and psycho-active agents.
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PMID:Differences in the structural requirements for selective interaction with neutral metalloendopeptidase (enkephalinase) or angiotensin-converting enzyme. Molecular investigation by use of new thiol inhibitors. 632 Nov 77

A thermolysin-like metalloendopeptidase, optimally active at a neutral pH, was identified in human serum. The enzyme cleaves the synthetic substrate glutaryl-Ala-Ala-Phe-2-naphthylamide at the Ala-Phe bond. Activity was determined by measuring the rate of formation of Phe-2-naphthylamide in a coupled enzyme assay in the presence of excess aminopeptidase M. 2-Naphthylamine released during the reaction was determined by a diazotization procedure. Enzyme activity is not affected by inhibitors of serine, thiol, or carboxyl proteases, but is sensitive to inhibition by metal chelators such as EDTA and o-phenanthroline. Dialysis against EDTA leads to loss of activity, which can be fully restored by zinc and cobalt ions. The serum enzyme closely resembles a membrane-bound metalloendopeptidase (EC 3.4.24.11) abundant in lung, spleen, and kidney in that both enzymes are inhibited by the same active-site-directed inhibitors. In addition, an antiserum obtained against the metalloendopeptidase from rabbit kidney shows strong cross-reactivity with the serum enzyme. Metalloendopeptidase activity was measured in 150 controls and in 95 patients with sarcoidosis; the two groups had significantly different enzyme activities (p less than 0.001). The mean enzyme activity in the sarcoidosis group was more than threefold higher than that of the control group. The mean enzyme activity for patients with active disease was more than double that of patients with inactive disease and more than four times that of controls (p less than 0.001). This is noteworthy because angiotensin converting enzyme, a zinc-dipeptidyl carboxypeptidase with a mechanism of action similar to that of the metalloendopeptidase, has also been reported to be increased in the serum of patients with active sarcoidosis. Enzyme activity in patients with active tuberculosis, primary pulmonary neoplasms, and idiopathic interstitial pulmonary fibrosis did not differ significantly from that of controls.
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PMID:Identification of a thermolysin-like metalloendopeptidase in serum: activity in normal subjects and in patients with sarcoidosis. 636 93

Rat brain neutral endopeptidase ("Enkephalinase") was shown to hydrolyze a series of fluorogenic substrates of the general structure 2-aminobenzoyl-(amino acid)n- leucylalanylglycine -4- nitrobenzylamide . The hydrolysis of these substrates was competitively inhibited by Leu5-enkephalin, demonstrating that these are indeed substrates for the rat brain neutral endopeptidase. Cleavage of the fluorogenic substrates yielded leucylalanylglycine -4- nitrobenzylamide as a common product. In addition, a series of inhibitors previously shown to inhibit thermolysin-like enzymes inhibited the hydrolysis of both Leu5-enkephalin and the synthetic substrates. The results of this study (a) demonstrate that the enkephalin-degrading endopeptidase is similar in specificity to thermolysin, (b) provide a continuous sensitive assay system for the enzyme, and (c) point out the potential use of this substrate class for probing the specificity of the enzyme.
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PMID:Fluorogenic substrates for the enkephalin-degrading neutral endopeptidase (Enkephalinase). 637 73

Interactive computer graphics was used as a tool in studying the cleavage mechanism of the model substrate Z-Phe-Phe-Leu-Trp by the zinc endopeptidase thermolysin. Two Michaelis complexes and three binding orientations of the tetrahedral intermediate to the crystal structure of thermolysin were investigated. Our results indicate that a Michaelis complex, which does not involve coordination of the scissile peptide to the zinc, is consistent with available experimental data and the most plausible of the two complexes. A tetrahedral intermediate complex wherein the two oxygens of the hydrated scissile peptide straddle the zinc in a bidentate fashion results in the most favorable interactions with the active site. The preferred tetrahedral intermediate and Michaelis complex provide a rationalization for the published substrate data. A trajectory for proceeding from the Michaelis complex to the tetrahedral intermediate is proposed. This trajectory involves a simultaneous activation of the zinc-bound water molecule concurrent with attack on the scissile peptide. A detailed ordered product release mechanism is also presented. These studies suggest some modifications and a number of extensions to the mechanism proposed earlier [Kester, W. R., & Matthews, B. W. (1977) Biochemistry 16, 2506; Holmes, M. A., & Matthews, B. W. (1981) Biochemistry 20, 6912]. The binding mode of the thermolysin inhibitor N-(1-carboxy-3-phenylpropyl)-L-leucyl-L-tryptophan [Monzingo, A. F., & Matthews, B. W. (1984) Biochemistry (preceding paper in this issue)] is compared with that of the preferred tetrahedral intermediate, providing insight into this inhibitor design.
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PMID:An interactive computer graphics study of thermolysin-catalyzed peptide cleavage and inhibition by N-carboxymethyl dipeptides. 652 36

The endopeptidase activity of mesophilic streptococci was characterized further by investigating the specificity of an intracellular endopeptidase from Streptococcus diacetylactis for beta-casein, derived peptides, and bradykinin. The inhibitory action of phosphoramidon as well as direct determinations of metal content showed this enzyme was a metalloprotein. Hydrolysis of native beta-casein was relatively low. Peptides obtained from the fraction soluble at pH 4.6 led to the demonstration that Pro186-Ile187 and Ala189-Phe190 were hydrolyzed by the enzyme. Two peptides derived from beta-casein by the action of chymosin were hydrolyzed efficiently: we observed hydrolysis of Lys176-Ala177, Lys169-Val170, and Pro206-Ile207. The Pro7-Phe8 bond of bradykinin was hydrolyzed rapidly, showing that this enzyme was efficient for the hydrolysis of prolyl peptide bonds. The protease was slightly less sensitive to phosphoramidon than was thermolysin. Metal analyses showed the enzyme contained 580 microgram of zinc and 4,760 microgram of calcium per gram protein. This protease is thus a true metalloenzyme (E.C.3.4.24.4), and its action may complete the hydrolysis initiated by chymosin remaining active in cheese curd by hydrolyzing peptides released by chymosin.
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PMID:Hydrolysis of beta-casein and peptides by intracellular neutral protease of Streptococcus diacetylactis. 676 75

Assay of solubilized dog pancreas microsomes revealed the presence of an endopeptidase which hydrolyzed the fluorogenic peptide substrate Suc-Ala-Ala-Phe-7-amino-4-methylcoumarin (AMC) between the alanine and phenylalanine positions. This activity was inhibited by phosphoramidon, 1,10-phenanthroline, and a number of synthetic inhibitors of thermolysin indicating that the enzyme is a zinc metallopeptidase. Endopeptidase activity was not inhibited by the serine protease inhibitors elastatinal, antipain, leupeptin, N-carbobenzyloxy-L-phenylethyl chloromethyl ketone, L-tosylamido-2-lysyethyl chloromethyl ketone, L-tosylamido-2-phenylethyl chloromethyl ketone, phenyl-methanesulfonyl fluoride, or low levels of chymostatin. The endopeptidase had a pH optimum between 7.0 and 7.5. The enzyme also hydrolyzed Suc-Ala-Ala-Ala-AMC and Suc-Ala-Gly-Ala-AMC in an analogous way to yield Ala-AMC. Thermolysis hydrolyzed Suc-Ala-Ala-Phe-AMC in an analogous way to the endopeptidase. However, thermolysin did not hydrolyze Suc-Ala-Ala-Ala-AMC or Suc-Ala-Gly-Ala-AMC, demonstrating that its substrate specificity differs from the endopeptidase.
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PMID:A zinc metalloendopeptidase associated with dog pancreatic membranes. 698 19


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