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)

Neutral endopeptidase 24.11 contains an active site arginine believed to function in substrate binding. This arginine is thought to form an ionic interaction with the COOH-terminal carboxylate of NEP substrates. The functionality of arginine 102 has been investigated by using site-directed mutagenesis to produce mutants in which this residue was converted to a lysine, glycine, glutamine, or glutamate. All of the mutants exhibited essentially full activity as determined with a synthetic peptide amide, glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide. In contrast, activity was detected only with the wild-type enzyme and the lysine mutant using a synthetic substrate containing a free COOH-terminal carboxylate, dansyl-Gly-Trp-Gly. Inhibition studies with the physiologically active peptide substrates substance P, endothelin, and angiotensin I, as well as substance P free acid, [D-Ala2,Leu5]enkephalin, and [D-Ala2,Leu5]enkephalinamide indicated a lack of importance of arginine 102 in substrate binding. With [D-Ala2,Met5]enkephalin and the chemotactic peptide, N-formyl-Met-Leu-Phe, a significant decrease in affinity is observed with the arginine 102 mutants. These results suggest that the contribution of arginine 102 to substrate binding is dependent upon the strength of other subsite interactions. Examination of dipeptides as inhibitors indicates that the nature and orientation of the P'2 residue is important in determining the strength of the interaction of arginine 102 with its substrates.
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PMID:Analysis of the importance of arginine 102 in neutral endopeptidase (enkephalinase) catalysis. 137 21

Aside from serving as zinc ligands, kinetic data has implicated one or more additional histidines as catalytic residues in neutral endopeptidase ("enkephalinase") action. One of these histidines has previously been identified as histidine 704 (Bateman et al., J. Biol. Chem., 265:8365-8368, 1990). In order to determine whether a second histidine is involved in catalysis each of these residues not previously changed have been converted to glutamine by site directed mutagenesis. The resultant recombinant enzymes possess full catalytic activity indicating that histidine 704 is the only catalytic histidine in the enzyme.
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PMID:Mutational analysis reveals only one catalytic histidine in neutral endopeptidase ("enkephalinase"). 157 57

An immortalized hypothalamic neuronal cell line was recently developed by genetically targeting the expression of the simian virus-40 large T-antigen in LHRH neurons. These GT1 cells were subcloned to GT1-1, GT1-3, and GT1-7 cells, and they have been shown to express the mRNA for pro-LHRH and secrete LHRH-like immunoreactive (IR) materials into the media. The purpose of our study was to biochemically and immunologically characterize the IR materials within and secreted from these cells. Both LHRH- and GnRH-associated peptide (GAP)-like IR materials were present and were secreted from these four cell lines. Up to 3% of the total cellular protein was composed of LHRH and GAP materials. When materials from the cell lysate and media were separated according to mol wt (Mr), at least three different pro-LHRH species were detected. These precursors contained both LHRH- and GAP-like IR determinants, and they eluted in the void volume and at approximately 10,000-12,000 and 8,400-8,500 Mr. A material that contained GAP-like IR eluted at approximately 6,500-6,800 Mr. This species is probably mouse GAP-(1-56) because it eluted on a reverse phase column in the approximate position of rat GAP-(1-56). Cell lysates contained a single LHRH-like IR form which coeluted on a size-exclusion column with synthetic LHRH. This material stimulated secretion of LH from anterior pituitary cells in a dose-response manner. By comparison, two different molecular forms of LHRH were detected in media at approximately 1,500 and 540 Mr. HPLC analyses revealed these peaks to be heterogeneous and to contain at least (Gln1)LHRH-(Gly11,Lys12,Arg13), (Gln1)LHRH-(Gly1,Lys12), LHRH-(Gly11), and LHRH. These experiments demonstrate that the cells contain and secrete multiple molecular forms of the pro-LHRH and that processing of the prohormone must involve 1) cleavage by an endopeptidase to give GAP-(1-56) and a C-terminally extended LHRH, 2) removal of C-terminal basic amino acids by a carboxypeptidase, 3) amidation of LHRH-(Gly11) to LHRH, and 4) cyclization of glutamine to pyroglutamate at the N-terminal of LHRH. These results provide the first evidence for intermediates in the metabolic pathway of pro-LHRH to LHRH.
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PMID:Metabolism of pro-luteinizing hormone-releasing hormone in immortalized hypothalamic neurons. 171 37

Neutral endopeptidase 24.11 is rapidly inactivated by N-bromoacetyl-D-leucylglycine in a reaction which follows first-order kinetics at pH 8 and 37 degrees C. The concentration dependence of inactivation revealed saturation kinetics with an apparent Ki of 10 mM and kappa inact of 0.4 min-1 at saturating inhibitor concentration. Enzyme can be protected from inactivation by either the substrate Leu5-enkephalin or the competitive inhibitors Phe-Gly or Phe-Ala. Inactivation of enzyme by N-bromo-[14C]acetyl-D-leucylglycine proceeds with the incorporation of a stoichiometric amount of labeled inhibitor. Tryptic digestion of the radioactively labeled enzyme followed by high performance liquid chromatography allowed the isolation of a modified peptide with the sequence T-D-V-H-S-P-G-N-F-R in which histidine (His704) is the modified residue. Site-directed mutagenesis was used to generate a mutant form of the enzyme in which histidine 704 was converted to a glutamine residue. This mutant enzyme retained less than 0.1% of the activity of the native enzyme. These results demonstrate that His704 is at the active site of neutral endopeptidase 24.11 and suggest a catalytic role for this residue.
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PMID:N-bromoacetyl-D-leucylglycine. An affinity label for neutral endopeptidase 24.11. 234 87

The primary mechanism of activation of intracellular prohormones seems to involve proteolytic cleavage at sequences of consecutive basic residues. Thus, all the known biologically active peptides derived from the prohormone of corticotropin and beta-endorphin appear to be excised initially by enzymes with this specificity. The C-terminal peptide, beta-endorphin (1-31), is generated by cleavage at a lysyl arginine sequence and an additional cleavage can give rise to the related peptides, beta-endorphin (1-27) and beta-endorphin (1-26). These derivatives of beta-endorphin are released by an endopeptidase that appears to catalyse cleavage on the carboxyl side of paired lysine residues, followed by the action of a carboxypeptidase B-like enzyme (Fig. 1). The beta-endorphin fragments, beta-endorphin (1-27) and beta-endorphin (1-26), have been isolated from porcine and bovine pituitary but the C-terminal dipeptide, glycyl glutamine, has not been reported previously. Here we describe the isolation of glycyl glutamine from porcine pituitary and present evidence for its presence in sheep brain stem. When applied ionophoretically to brain stem neurones in the rat, the dipeptide exhibited an inhibitory action on cell firing.
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PMID:Glycyl glutamine, an inhibitory neuropeptide derived from beta-endorphin. 631 48

Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle-associated membrane protein (VAMP). A synthetic peptide of human/rat VAMP-2 [VAMP-2-(60-94)] is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane-associated protein (at the Gln76-Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin. Cleavage of the VAMP-2 peptide was demonstrated by both botulinum type B neurotoxin (Km = 3.3 x 10(-4) M) and by its purified light subunit (Km = 3.5 x 10(-4) M). The endopeptidase displayed a pH optimum of 7.0-7.5 and was inhibited by greater than 0.2 M NaCl and greater than 0.05 M sodium phosphate. Neurotoxin which had been inactivated by dialysis against EDTA could be re-activated by incubation with various divalent cations, notably Zn2+ and Cu2+. The substrate specificity of botulinum type B neurotoxin was studied using various analogues of VAMP-2 (60-94). The neurotoxin cleaved selectively to the N-terminal side of phenylalanine and tyrosine; no activity was observed with either leucine, valine or alanine in the P'1 position. The properties of the P1 amino acid were less critical; the neurotoxin cleaving the C-terminus of glutamine, asparagine and alanine. A substrate analogue with valine in the P1 position corresponding to the sequence of rat VAMP-1 was not cleaved. The rate of cleavage of a substrate analogue representing the sequence of human VAMP-1, however, was more than twofold that of the VAMP-2 peptide. The properties and substrate specificity of botulinum type B neurotoxin suggest that the toxin represents a novel class of endopeptidase which requires a specific peptide substrate conformation for the expression of proteolytic activity.
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PMID:Peptide substrate specificity and properties of the zinc-endopeptidase activity of botulinum type B neurotoxin. 792 46

A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was cloned into the pQE expression vector, resulting in the fusion of a hexahistidine at the N-terminus. The recombinant enzyme was expressed to high levels in Escherichia coli (approximately 10 mg/liter of culture) and purified to homogeneity in a two-step procedure. A number of peptides were studied as substrates for the enzyme. The enzyme cleaves the following peptides at the Gly3-Phe4 bond: enkephalins, dynorphins A-6, A-8, A-9, A-10, A-13, and A-17, and alpha-neo-endorphin. In addition the enzyme hydrolyzes bradykinin, substance P, beta-endorphin, ACTH, and VIP. Although the cleavage patterns observed are similar to that seen with mammalian neutral endopeptidase, the lactococcal enzyme more efficiently cleaves larger peptide substrates. As observed with the mammalian neutral endopeptidase, the lactococcal enzyme exhibits higher kcat/K(m) values for the enkephalins than for their corresponding amides, indicating the functionality of an active-site arginine. Inactivation of the lactococcal endopeptidase by diethyl pyrocarbonate and protection afforded by the substrate dynorphin A-6 indicate the functionality of a positionally conserved active-site histidine. This was confirmed by demonstrating that conversion of this histidine, histidine 587, to glutamine generated inactive enzyme. Similarly, conversion of the putative zinc ligand glutamate 535 to glutamine led to inactive enzyme. These studies indicate a conservation of critical catalytic residues between the two enzymes and suggest that the lactococcal endopeptidase is a better model than thermolysin for the mammalian enzyme.
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PMID:Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (neprilysin). 880 62

Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S2 site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1-9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.
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PMID:Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley. 881 95

We have identified a preparation of recombinant murine interleukin-6 (mIL-6) that, in addition to the anticipated product, also contained approximately equal amounts of mIL-6 with a C-terminal pentapeptide extension. The extension mutant was generated by readthrough of the stopcodon, and termination at a second in-frame stopcodon 12 base pairs 3' in the expression vector. Aliquots of the preparation were subjected to proteolytic digestion with Asp-N and Lys-C-endopeptidase. The resultant peptides were separated by reversed-phase capillary HPLC, and analysed using a combination of mass spectrometry and N-terminal sequence analysis. These data revealed a C-terminal pentapeptide (Gln-Gly-Ser-Val-Asp) extension, with the authentic stopcodon being translated as glutamine. The extension mutant was isolated by reversed-phase HPLC and shown to have similar mitogenic activity to mIL-6 on murine hybridoma 7TD1 cells.
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PMID:Identification and characterization of recombinant murine interleukin-6 with a C-terminal pentapeptide extension using capillary reversed phase HPLC-MS and edman degradation. 941 11

Escherichia coli biotin carboxylase was affinity labeled with adenosine diphosphopyridoxal to identify its ATP binding site. Lysyl endopeptidase digestion of the modified protein, followed by high performance liquid chromatography separation and amino acid sequencing allowed to identify lysine-238 to be the site of modification. Site-directed mutagenesis of this residue into alanine, arginine or glutamine resulted in mutants with much decreased activity. Lysine-238 seems to interact with the gamma-phosphate group of ATP but is not involved in catalysis.
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PMID:Identification of lysine-238 of Escherichia coli biotin carboxylase as an ATP-binding residue. 963 61


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