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)

Among the five peptidase known to be located in the microvillus membrane of the renal proximal tubule are two enzymes with endopeptidase activity. Neutral endopeptidase, a zinc-dependent enzyme, has a broad specificity comparable to that of thermolysin, and like the latter may be specifically inhibited by phosphoramidon. Dipeptidyl peptidase IV, a serine enzyme, is very sensitive to inhibition by diisopropyl phosphorofluoridate. It is also capable of endopeptidase activity, hydrolysing bonds involving the carboxyl group of proline.
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PMID:Endopeptidases in the brush border of the kidney proximal tubule. 24 85

Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.
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PMID:Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11. 167 40

Neutral endopeptidase (EC 3.4.24.11) is a major constituent of kidney brush border membranes. It is also present in the brain where it has been shown to be involved in the inactivation of opioid peptides, methionine- and leucine-enkephalins. For this reason this enzyme is often called 'enkephalinase'. In order to characterize the primary structure of the enzyme, oligonucleotide probes were designed from partial amino acid sequences and used to isolate clones from kidney cDNA libraries. Sequencing of the cDNA inserts revealed the complete primary structure of the enzyme. Neutral endopeptidase consists of 750 amino acids. It contains a short N-terminal cytoplasmic domain (27 amino acids), a single membrane-spanning segment (23 amino acids) and an extracellular domain that comprises most of the protein mass. The comparison of the primary structure of neutral endopeptidase with that of thermolysin, a bacterial Zn-metallopeptidase, indicates that most of the amino acid residues involved in Zn coordination and catalytic activity in thermolysin are found within highly honmologous sequences in neutral endopeptidase.
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PMID:Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA. 244 Jun 77

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found in the plasma membrane of many cell types. The cDNA coding for the complete primary structure of neutral endopeptidase has recently been cloned and sequenced (Devault, A. Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322). Comparison of the sequence of neutral endopeptidase with that of thermolysin, a bacterial Zn-metalloendopeptidase, suggests that Glu-584 in neutral endopeptidase probably corresponds to Glu-143 in thermolysin, which is an essential amino acid involved in catalysis. To test directly the importance of Glu-584 in the catalytic activity of neutral endopeptidase by site-directed metagenesis, we have constructed an expression vector in which the rabbit kidney cDNA encoding the entire neutral endopeptidase sequence is introduced downstream from the SV40 virus early promotor. After transfection in COS-1 monkey kidney cells, this vector was found to promote the expression of a protein with biochemical and catalytic properties identical to kidney neutral endopeptidase. Oligonucleotide-directed mutagenesis of Glu-584 to either valine or aspartic acid completely abolished the enzymatic activity of the recombinant protein without changing its affinity for the substrate-related tritiated inhibitor [3H]N-[(2R,2S)-3-hydroxyamino-carbonyl-2-benzyl-1-oxopropyl]-glycine. This observation clearly identifies Glu-584 as one of the important residues responsible for the catalytic activity of the enzyme.
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PMID:Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site. 289 75

Neutral endopeptidase (NEP) is a membrane-bound mammalian ectopeptidase that contains a catalytic zinc ion in its active site. Previous studies showed that the active site, and especially the zinc-binding site of NEP, have features in common with the prototypical bacterial zinc protease, thermolysin. Sequence comparison reveals that both enzymes have a conserved Asp residue (Asp650 in NEP and Asp170 in thermolysin) located four positions on the C-side of the third zinc ligand. In thermolysin, this residue is involved in a carboxylate-histidine-zinc interaction whose functional role has never been established [Christianson, D. W. & Alexander, R. S. (1990) Nature 346, 225]. To test the hypothesis that, in NEP, this residue is important for catalysis, we have changed Asp650 of NEP by site-directed mutagenesis and expressed the mutant enzymes in COS-1 cells. Substitution of Glu, Asn or Ala for Asp650 resulted in mutant enzymes exhibiting drastic decreases in specific activity. Binding experiments using the zinc-chelating inhibitor [3H]-N-[(2RS)-4-(hydroxyamino)-1,4-dioxo-2-(phenylmethyl)butyl]glycine suggested that the zinc ion is present in the active site of these mutant enzymes. These results strongly support the conclusion that Asp650 in NEP is crucial for hydrolytic activity.
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PMID:Asp650 is crucial for catalytic activity of neutral endopeptidase 24-11. 816 35

Mammalian cell-surface peptidases participate in the postsecretory processing and metabolism of neuropeptides and peptide hormones. Neutral endopeptidase-24.11 (NEP) is the prototype of a family of zinc metallopeptidases that also includes the endothelin-converting enzymes (ECE) and which are structurally related to the bacterial enzymes thermolysin and lactococcal endopeptidase. Two other mammalian gene products exhibit strong homology with NEP: the erythrocyte cell-surface antigen, KELL; and the putative product of the PEX gene, which has been associated with X-linked hypophosphatemic rickets. No enzymic activity has yet been attributed to KELL and PEX proteins, and they remain peptidases in search of a substrate. A wide range of biologically active peptide substrates has been described for NEP, of which the enkephalins and the atrial natriuretic peptide family have assumed greatest significance. Endothelin-converting enzyme catalyses the final step in the biosynthesis of the vasoconstrictor peptide, endothelin (ET). Like NEP, it is a type II integral membrane protein, but is expressed predominantly in endothelial cells. Isoforms of ECE (ECE-1alpha, ECE-1beta, and ECE-2) exist that differ in a number of characteristics. In particular, ECE-1, through the paracrine effects of ET-1, may contribute to the proliferation of smooth muscle after angioplasty and to the development of human atherosclerosis. Inhibitors of ECE and NEP may have important therapeutic applications in cardiovascular and renal medicine.
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PMID:Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX. 914 2