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)

A porcine kidney microsomal metalloendopeptidase has been enriched 3900-fold. Gel filtration on a calibrated Toyo-Soda G-3000 SW column indicated an appropriate molecular weight for the endopeptidase of 88,000 +/- 2000. The purified enzyme is inhibited by a number of synthetic inhibitors of thermolysin. The endopeptidase hydrolyzes the succinyl (Suc)-containing fluorogenic peptide substrate Suc-Ala-Ala-Phe-(7-amino-4-methylcoumarin) at the Ala-Phe position with a Km of 2.9 X 10(-4) M. The endopeptidase also hydrolyzes a variety of peptides including corticotropin, substance P, angiotensin I and II, neurotensin, somatostatin, bradykinin, and the renin tetradecapeptide substrate. The endopeptidase hydrolyzes both [Leu]- and [Met]enkephalin at the Gly-Phe bond.
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PMID:Purification of a membrane-bound metalloendopeptidase from porcine kidney that degrades peptide hormones. 703 58

Neprilysin (EC 3.4.24.11) is a Zn2+ metallopeptidase involved in the degradation of biologically active peptides, e.g. enkephalins and atrial natriuretic peptide. The substrate specificity and catalytic activity of neprilysin resemble those of thermolysin, a crystallized bacterial Zn2+ metalloprotease. Despite little overall homology between the primary structures of thermolysin and neprilysin, many of the amino acid residues involved in catalysis, as well as Zn2+ and substrate binding, are highly conserved. Most of the active-site residues of neprilysin have their homologues in thermolysin and have been characterized by site-directed mutagenesis. Furthermore, hydrophobic cluster analysis has revealed some other analogies between the neprilysin and thermolysin sequences [Benchetrit, Bissery, Mornon, Devault, Crine and Roques (1988) Biochemistry 27, 592-596]. According to this analysis the role of Asn542 in the neprilysin active site is analogous to that of Asn112 of thermolysin, which is to bind the substrate. Site-directed mutagenesis was used to change Asn542 to Gly or Gln residues. The effect of these mutations on substrate catalysis and inhibitor binding was examined with a series of thiorphan-like compounds containing various degrees of methylation at the P2' residue. For both mutated enzymes, determination of kinetic parameters with [D-Ala2,Leu5]enkephalin as substrate showed that the large decrease in activity was attributable to an increase in Km (14-16-fold) whereas kcat values were only slightly affected (2-3-fold decrease). This is in agreement with Asn542 being involved in substrate binding rather than directly in catalysis. Finally, the IC50 values for thiorphan and substituted thiorphans strongly suggest that Asn542 of neprilysin binds the substrate on the amino side of the P2' residue by formation of a unique hydrogen bond.
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PMID:Evidence that Asn542 of neprilysin (EC 3.4.24.11) is involved in binding of the P2' residue of substrates and inhibitors. 748 5

1. Phosphoramidon, a potent inhibitor of endopeptidase-24.11 (E-24.11) and thermolysin, has been shown to reduce the hypertensive effect of exogenous big endothelin-1 (big ET-1) in rats. To examine whether E-24.11 or thermolysin convert big ET-1 to endothelin-1 (ET-1) and C-terminal fragment (CTF), the effects on porcine and human big ET-1 of each of the purified enzymes were compared in vitro. 2. For E-24.11, the relative rates of hydrolysis were ET-1 > CTF >> big ET-1. The relative half-lives for hydrolysis of 3 nmol of each peptide by 200 ng enzyme were: big ET-1 > 24 h; ET-1, 37 min; CTF, 57 min. For comparison, the half-life for hydrolysis of substance P under similar conditions was 2.1 min. 3. For thermolysin the relative rates of hydrolysis were found to be big ET-1 > CTF > ET-1. The relative half-lives for hydrolysis of 3 nmol peptide by 50 ng enzyme were: big ET-1, 25 min; ET-1, 56 min; CTF, 47 min. 4. Because the low rate of conversion of big ET-1 to ET-1 by E-24.11 did not yield sufficient ET-1 for h.p.l.c. quantification a RIA specific for ET-1(16-21) was used to study further the hydrolysis of big ET-1 by E-24.11. Incubation of big ET-1 (0.2-2 nmol) with E-24.11 (4-400 ng) generated ET-1 levels of between 1.7 and 33 pmol measured by RIA. Incubation of big ET-1 (2 nmol) with E-24.11 (40 ng) for 8 h showed that steady state levels of ET-1 were achieved after 4 h indicating that the rate of ET-1 degradation was then equal to the formation of new ET-1. Characterization of the immunoreactivity by h.p.l.c. and RIA confirmed that authentic ET-1 had been produced, but the yield was insufficient for verification by mass spectrometry.5. Both ET-l-like and CTF-like peaks were detected at 214 nm when the products of big ET-1 hydrolysis by thermolysin were resolved by h.p.l.c. RIA and mass spectrometry confirmed the production of ET-1 with amounts in the range 120-160 pmol.6. The hydrolysis profile of ET-1 by E-24.11 and thermolysin shows that both enzymes have some common cleavage sites consistent with their similar specificities hydrolysing on the amino side of a hydrophobic residue.7. Thermolysin, for which 3D structural information is available, may represent a better model for endothelin converting enzyme (ECE) action than E-24.11 and could be useful for the design of ECE inhibitors. Since E-24.11 can both synthesize and hydrolyse ET-1, the presence of E-24.11 in membrane fractions or in partially purified ECE preparations may produce misleading estimates of ECE activity.
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PMID:Generation by the phosphoramidon-sensitive peptidases, endopeptidase-24.11 and thermolysin, of endothelin-1 and c-terminal fragment from big endothelin-1. 752 8

The reaction of neprilysin and thermolysin with a series of cyclic beta-turn peptides, varying in length from 6 to 14 residues, has been studied. All of the cyclic peptides bind to neprilysin with their affinity increasing from 113 microM for the 6-membered ring to 17 microM for the 14-membered ring. The 6-membered cyclic peptide was not hydrolyzed. However, kcat increased from 1.5 min-1 for the 8-membered cyclic peptide to 148 min-1 for the 14-membered cyclic peptide. With thermolysin binding of the 6- or 8-membered cyclic peptides was not detected. The Km values for the 10-, 12-, and 14-membered cyclic peptides were all in the 100 microM range. With thermolysin, kcat increased from 7 min-1 for the 10-membered cyclic peptide to 27,000 min-1 for the 14-membered cyclic peptide. Cyclic peptides were all cleaved at N-terminally directed sites. Modeling of the binding of a cyclic peptide, structurally similar to the 12-membered cyclic beta-turn peptide described above, into the active site of thermolysin shows that only half of the substrate makes contact with the enzyme and that only residues on one side of the peptide could fit into the active site. From these studies it is concluded that key factors which influence catalysis include not only peptide sequence, but the flexibility of the peptide and the orientation of the S'1 residue in a cyclic peptide.
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PMID:Reaction of neprilysin (neutral endopeptidase) and thermolysin with cyclic peptides. 757 14

The ligand-filled 32-kDa fragment of the porcine estradiol receptor extending from His267 to the C-terminal Ile595 was purified to homogeneity by adsorption to mAb 13H2. The native protein was exposed at 4 degrees C to a panel of proteases: thermolysin, subtilisin, pronase, elastase, ficin, bromelain, endopeptidase Lys-C, both in the dimer and the monomer state, and chymotrypsin at pH 8.2 only. The digests were analysed by SDS/PAGE/Western blotting for Coomassie staining and immunostaining. Peptides were sequenced from blots. The majority of cleavage sites in upper domain E (8 out of 11) amassed in the Leu296-Leu310 stretch. Cleavage at Leu319 was seen with subtilisin and at Tyr328 with chymotrypsin. Susceptability to enzymic proteolysis was also pronounced in Thr465-Glu470 at the center of domain E. Three peptides, 13 kDa with thermolysin, beginning at Leu337, 6 kDa and, in low yield, 5 kDa with endopeptidase Lys-C beginning at Asp473 resp. Cys417 were only obtained from the monomer substrate. The various digests featured either 27-23-kDa peptides or mixtures of 17-13-kDa and 12-7-kDa peptides separable by SDS/PAGE. All peptides with N-termini between Leu297 and Ser329 reacted with mAb 13H2. The digests showed high peaks of bound estradiol in the dimer position of 32-kDa fragment controls on density gradient centrifugation at pH 7.4. However, the property of proton-driven dissociation was only preserved in the pronase, elastase and chymotrypsin digests with peptides extending beyond the His547-ArgLeuHis550 motif. The preservation of the estradiol-binding niche in the tightly complexed peptides of domain E was also demonstrated by refilling after steroid removal. The sites exposed to proteolytic enzymes and the epitope for 13H2 attachment are in good agreement with surface probability plots.
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PMID:Surface mapping of the ligand-filled C-terminal half of the porcine estradiol receptor by restricted proteolysis. 763 63

Raw-starch-digesting amylase (RSDA) is a key extracellular enzyme of mesophilic Bacillus circulans F-2 which uses raw starch granules as a carbon source. Previous work has demonstrated that there are two domains of the enzyme during digestion with subtilisin, and that RSDA activity is selectively inactivated by limited proteolysis with subtilisin, which cleaves the enzyme between these hydrolytic and adsorption domains (Kim, C.-H., Kwon, S.-T., Taniguchi, H. and Lee, D.-S. (1992) Biochim. Biophys. Acta 1122, 243-250). In this work we show that a similar phenomenon is observed during limited proteinase K, thermolysin and endopeptidase Glu-C proteolysis of the enzyme. Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA. The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63 and 30-kDa fragments. Similar patterns were obtained with endopeptidase Glu-C or thermolysin. All proteolytic digests contained a common, major 63-kDa fragment. Inactivation of RSDA activity results from splitting off the C-terminal domain. Hence, it seems probable that the proteinase-sensitive locus is in a hinge region susceptible to cleavage. Extracellular enzymes immunoreactive towards anti-RSDA were detected through whole bacterial cultivation. 93, 75, 63, 55, 38 and 31-kDa proteins were immunologically identical to RSDA. Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin. These results suggest that enzyme heterogeneity of the raw-starch hydrolysis system might arise from the endogenous proteolytic activity of the bacterium.
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PMID:Domain structure and multiplicity of raw-starch-digesting amylase from Bacillus circulans: extensive proteolysis with proteinase K, endopeptidase Glu-C and thermolysin. 769 Nov 84

Tetanus neurotoxin (TeNT) blocks neuroexocytosis via a zinc-endopeptidase activity highly specific for vescicle-associated membrane protein(VAMP)/synaptobrevin. TeNT is the prototype of clostridial neurotoxins, a new family of metalloproteinases. They consist of three domains and the proteolytic activity is displayed by the 50-kDa light chain (L chain). The L chain was isolated here in the native state from bacterial filtrates of Clostridium tetani and its structure was studied via circular dichroism (CD) and fluorescence spectroscopy. The secondary structure content (27% alpha-helix and 43% beta-sheet), estimated by far-ultraviolet CD measurements, was in reasonable agreement with that obtained by standard predictive methods (25% alpha-helix and 49% beta-sheet). Moreover, the hypothetical zinc-binding motif, encompassing residues His-Glu-Leu-Ile-His, was correctly predicted to be in alpha-helical conformation, as also expected on the basis of the geometrical requirements for a correct coordination of the zinc ion. Both near-ultraviolet CD and fluorescence data strongly suggest that the single Trp43 residue is buried and constrained in a hydrophobic environment, likely distant from the zinc ion located in the active-site cleft. The contribution of the bound zinc ion to the overall conformation of TeNT L chain was investigated by different and complementary techniques, including spectroscopic (far- and near-ultraviolet CD, fluorescence, second derivative absorption spectroscopy) as well as proteolytic probes. The results indicate that the zinc ion plays little, if any, role in determining the structural properties of the L chain molecule. Similarly, the metal-free apo-enzyme and the holo-protein share common stability features evaluated in respect to different physico-chemical parameters (pH, temperature and urea concentration). These results parallel those obtained on thermolysin, a zinc-dependent neutral endoprotease from Bacillus thermoproteolyticus, where both conformational and stability properties are unchanged upon zinc removal.
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PMID:Structural studies on the zinc-endopeptidase light chain of tetanus neurotoxin. 774 50

1. Adamalysin II, alias proteinase II, a 24-kDa zinc-endopeptidase from the snake venom of Crotalus adamanteus, is a member of a large family of metalloproteinases isolated as small proteinases or proteolytic domains of mosaic hemorrhagic proteins from various snake venoms. Homologous domains have been recently detected in multimodular mammalian reproductive tract proteins and in mammalian gene products, somatic rearrangements of which seem to be linked to primary breast cancers. 2. The 2.0 A X-ray crystal structure of adamalysin II reveals an ellipsoidal molecule with a shallow active-site cleft separating a relatively irregularly folded sub-domain from the main molecular body composed of a 5-stranded beta-sheet and four alpha-helices. Opposite to this active-site cleft is an integrated calcium ion liganded by carbonyl and strongly conserved carboxylate/carboxamide residues. The folding of the peptide fragment containing the zinc-binding motif HExxHxxGxxH bears only a distant resemblance to thermolysin; it is identical to that found in astacin, in collagenases, and in serralysins, with the three histidines (His142, His146, His152) and a water molecule (linked to the glutamic acid Glu143) likewise constituting the zinc ligand; similar to collagenases, but in contrast to astacin, adamalysin II lacks a fifth (tyrosine) zinc ligand, leaving its zinc-ion tetrahedrally coordinated. Furthermore, adamalysin II shares an identical active-site basement formed by a common Met-turn. 3. Due to their virtually identical active-site environment and similar folding topology, the snake venom metalloproteinases (hitherto called adamalysins) and the three other proteinases might be grouped into a common superfamily called metzincins with distinct differences from the thermolysin family.
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PMID:The crystal structure of adamalysin II, a zinc-endopeptidase from the snake venom of the eastern diamondback rattlesnake Crotalus adamanteus. 774 94

Hydroxamic acids 6a-h, derived from malonyl amino acids, and 25a-d, derived from succinyl amino acids, were synthesized as inhibitors of human bronchiolar smooth muscle endothelin-converting enzyme (HBSM ECE). Several unexpected side reactions were discovered, particularly in the synthesis of hydroxamates derived from succinates. In vitro evaluation against human bronchiolar ECE revealed that in all cases hydroxamates derived from malonate were more potent than hydroxamates derived from succinate. Isopropyl and isobutyl P1' side chains were suitable; omission of the P1' side chain seriously diminished potency. In the P2' position, several amino acids gave potent malonate-derived hydroxamate inhibitors (6b, d-h, IC50 = 0.2-6.8 nM), and beta-Ala provided an extremely potent inhibitor (6c, IC50 = 0.01 nM). C-terminus carboxylates are much more potent ECE inhibitors than the corresponding amides. Most of the hydroxamates were also potent inhibitors of thermolysin and neutral endopeptidase (NEP); however, the P2' beta-Ala derivative 6c uniquely inhibited HBSM ECE much more potently than NEP.
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PMID:Hydroxamic acids as potent inhibitors of endothelin-converting enzyme from human bronchiolar smooth muscle. 778 43

Zinc endopeptidase thermolysin can be inhibited by a series of phosphorus-containing peptide analogues, Cbz-Gly-psi (PO2)-X-Leu-Y-R (ZGp(X)L(y)R), where X = NH, O, or CH2; Y = NH or O; R = Leu, Ala, Gly, Phe, H, or CH3. The affinity correlation as well as an X-ray crystallography study suggest that these inhibitors bind to thermolysin in an identical mode. In this work, we calculate the electrostatic binding free energies for a series of 13 phosphorus-containing inhibitors with modifications at X, Y, and R moieties using finite difference solution to the Poisson-Boltzmann equation. A method has been developed to include the solvation entropy changes due to binding different ligands to a macromolecule. We demonstrate that the electrostatic energy and empirically derived solvation entropy can account for most of the binding energy differences in this series. By analyzing the binding contribution from individual residues, we show that the energy of a hydrogen bond is not confined to the donor and acceptor. In particular, the positive charges on Zn and Arg 203, which are not the acceptors, contribute significantly to the hydrogen bonds between two amides of ZGpLL and the thermolysin.
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PMID:Binding of phosphorus-containing inhibitors to thermolysin studied by the Poisson-Boltzmann method. 779 20


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