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

Neutral endopeptidase (EC 3.4.24.11, NEP) is a Zn-metallopeptidase involved in the degradation of biologically active peptides, notably the enkephalins and atrial natriuretic peptide. Recently, the structure of the active site of this enzyme has been probed by site-directed mutagenesis, and 4 amino acid residues have been identified, namely 2 histidines (His583 and His587), which act as zinc-binding ligands, a glutamate (Glu584) involved in catalysis, and an arginine residue (Arg102), suggested to participate in substrate binding. Site-directed mutagenesis has now been used to investigate the role of 4 other arginine residues (Arg408, Arg409, Arg659, and Arg747) that have been proposed as possible active site residues and to further analyze the role of Arg102. In each case, the arginine was replaced with a methionine, and both enzymatic activity and the IC50 values of several NEP inhibitors were measured for the mutated enzymes and compared to wild-type enzyme. The results suggest that 2 arginines, Arg102 and Arg747, could both be important for substrate and inhibitor binding. Arg747 seems to be positioned to interact with the carbonyl amide group of the P'1 residue and can be modified when the enzyme is treated with the arginine-specific reagents phenylglyoxal and butanedione. Arg102 could be positioned to interact with the free carboxyl group of a P'2 residue in some substrates and inhibitors and can be modified by phenylglyoxal but not by butanedione. The results could explain the dual dipeptidylcarboxypeptidase and endopeptidase nature of NEP.
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PMID:Evidence that both arginine 102 and arginine 747 are involved in substrate binding to neutral endopeptidase (EC 3.4.24.11). 198 94

gamma-Glutamyl hydrolase is a ubiquitous enzyme that has the capacity to cleave gamma-glutamyl bonds of cellular folyl- and antifolylpoly-gamma-glutamates. This study has revealed that the enzyme is secreted by primary cultures of rat hepatocytes and by H35 hepatoma cells. It was found that more than 99% of the total enzyme from H35 cells accumulated in the medium after 48 hr incubation with the serum-free medium. The cells were shown to remain intact during the secretion period since lactate dehydrogenase, dihydrofolate reductase and lysosomal hydrolases other than gamma-glutamyl hydrolase were retained within the cell. When PteGlu5 (folylGlu4) is used as a substrate the initial product is PteGlu (folate), and there is no appearance of intermediate chain length pteroyl polyglutamates. Therefore, the secreted and cellular gamma-glutamyl hydrolase from hepatoma cells appears to be an endopeptidase. Polyclonal antibodies to the poly-gamma-glutamate substrates of the enzyme were prepared and characterized. The antibodies recognize the structural differences between alpha- and gamma-glutamyl linkages but appear equally active with PteGlu5 and its analogs such as 4-NH2-10-CH3PteGlu5 and pABAGlu5. The affinity of the antibodies is related to the gamma-glutamyl structure since L-glutamic acid, folate or p-aminobenzoic acid are inactive with the antibodies. Furthermore, poly-gamma-glutamate has lower affinity for the antibodies than the poly-gamma-glutamate derivatives of PteGlu, 4-NH2-10-CH3PteGlu or pABA.
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PMID:The properties and function of gamma-glutamyl hydrolase and poly-gamma-glutamate. 768 89

gamma-Glutamyl hydrolase has been partially purified and characterized from conditioned culture medium of H35 hepatoma cells. Evidence for heterogeneity of the enzyme is derived from its elution as three distinct peaks of enzymatic activity when the enzyme is purified by TSK-butyl-Sepharose column chromatography. These three enzyme fractions appear to have identical catalytic properties but, as yet, the basis for their resolution is not understood. A rapid, sensitive and simple assay based on reverse-phase HPLC fluorescent detection with pre-column derivatization using o-phthalaldehyde (OPA) was developed to separate OPA-derivatives of poly-gamma-glutamates and glutamic acid. Using this assay and the standard HPLC assay for pteroylpolyglutamates, the enzyme appears to be an endopeptidase with respect to pteroylpenta-gamma-glutamate (PteGlu5), methotrexate penta-gamma-glutamate (4-NH2-10-CH3PteGlu5) and p-aminobenzoyl-penta-gamma-glutamate (pABAGlu5). The initial products are PteGlu1 (or 4-NH2-10-CH3PteGlu1 or pABAGlu1) and intact tetra-gamma-glutamate, which is subsequently degraded to glutamic acid. When penta-gamma-glutamate is the substrate, the cleavage of the gamma-bonds by the enzyme is less ordered, with the early appearance of mono-, di-, tri- and tetraglutamate. Poly-alpha-glutamate is not a substrate nor are pABA-gamma-Glu5 or penta-gamma-glutamate covalently linked to albumin. 4-NH2-10-CH3PteGlu2 or Glu5 bound to dihydrofolate reductase is not a substrate for the enzyme, offering further evidence that protein-associated poly-gamma-glutamates are poor substrates for gamma-glutamyl hydrolase from H35 hepatoma cells.
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PMID:The properties of the secreted gamma-glutamyl hydrolases from H35 hepatoma cells. 834 22

We present a model for the three-dimensional structure of the glutamate-specific endopeptidase from Streptomyces griseus based on the crystal structures of other bacterial proteases of the trypsin family. For the first time a structural model is described which attempts to explain the basis of P1 glutamate specificity in serine proteases. Several important changes to the S1 pocket with respect to other members of the family of different specificity are described. Of particular interest is the presence of a histidine at position 213 and the substitution of Arg-138 by lysine. Other biochemical evidence concerning substrate preferences can be rationalized on the basis of the model.
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PMID:A structural model for the glutamate-specific endopeptidase from Streptomyces griseus that explains substrate specificity. 850 58

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

This study examined the effects of substance P (SP) and calcitonin gene-related peptide (CGRP) on synaptic transmission in a pontine slice containing the parabrachial nucleus (PBN). Stimulation of the ventral, external lateral portion of the PBN elicited glutamate-mediated EPSCs in cells recorded using the nystatin perforated-patch recording technique in the external lateral, external medial, and central lateral subnuclei of the PBN. Bath application of SP or CGRP dose-dependently and reversibly attenuated the evoked EPSC. The attenuation of the EPSC induced by both of these peptides was not accompanied by changes in input resistance of PBN cells over a wide voltage range, nor did these peptides alter the inward current induced by a brief bath application of AMPA. The combined application of subthreshold concentrations of these peptides revealed a synergistic interaction in reducing the evoked EPSC. The substance P neurokinin-1 receptor antagonist CGP49823 completely and reversibly blocked both the SP- and the CGRP-induced attenuation of the EPSC. However, the rat CGRP receptor antagonist human-CGRP8-37 did not block the actions of CGRP or SP on the EPSC. Using a metabolically stable analog of SP, SP (5-11), or an endopeptidase inhibitor, phosphoramidon, we were able to demonstrate that CGRP enhances the SP effect by inhibiting an SP endopeptidase. Application of phosphoramidon also revealed an endogenous SP "tone" apparently made effective by blockade of the endopeptidase. These results suggest that SP (and CGRP indirectly through an inhibition of the SP endopeptidase) acts on presynaptic NK-1 receptors to cause an inhibition of excitatory transmission in the PBN. These results indicate an important role of endopeptidases in regulating synaptic modulation by peptides.
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PMID:Peptidergic modulation of synaptic transmission in the parabrachial nucleus in vitro: importance of degradative enzymes in regulating synaptic efficacy. 881 87

The aim of the present study was to investigate some putative neurotransmitters involved in nociception and pain in parturients during active labour experiencing intense visceral pain. The concentration of the excitatory amino acid aspartate was significantly increased, and there was a tendency for an increase in glutamate, in lumbar cerebrospinal fluid (CSF) of parturients in active vaginal labour compared with control patients without pain subjected to elective caesarean section. The CSF concentration of the nitric oxide breakdown product nitrate was significantly decreased in parturients compared with control patients and healthy volunteers. No significant differences in the concentrations of substance P, substance P-endopeptidase or met-enkephalin were detected between parturients and controls. Our data suggest a paradoxical negative relationship between CSF concentrations of excitatory amino acids and nitric oxide in labour pain. The mechanisms behind this finding is unclear at present.
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PMID:Increased cerebrospinal fluid concentration of aspartate but decreased concentration of nitric oxide breakdown products in women experiencing visceral pain during active labour. 914 Oct 79

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM 12 is catalytically active. We used the trapping mechanism of alpha2-macroglobulin to assay for protease activity of wild-type and mutant ADAM 12 proteins produced in a COS cell transfection system. We found that ADAM 12 is synthesized as a zymogen, with the prodomain maintaining the metalloprotease in a latent form, probably by means of a cysteine switch. The zymogen could be activated chemically by alkylation with N-ethylmaleimide. Cleavage of the prodomain at a site for a furin-like endopeptidase resulted in an ADAM 12 protein with proteolytic activity. The protease activity was sensitive to inhibition by 1,10-phenanthroline and could be eliminated by mutation of the critical glutamate residue at the active site. The demonstration that the ADAM 12 metalloprotease domain is functional may have important implications for future studies that explore the role of ADAM 12 protein in development and disease.
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PMID:Human ADAM 12 (meltrin alpha) is an active metalloprotease. 964 63

Type A botulinum neurotoxin (BoNT/A) is a zinc endopeptidase that contains the consensus sequence HEXXH (residues 223-227) in the toxic light chain (LC). The X-ray structure of the toxin has predicted that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. The functional implication of E224 in the motif of LC was investigated by replacing the residue with glutamine and aspartate using site-directed mutagenesis. Substitution of Glu-224 with Gln (E224Q) resulted in a total loss of the endopeptidase activity, whereas substitution with Asp (E224D) retained about 1.4% of the enzymatic activity (k(cat) 140 vs 1.9 min(-1), respectively). However, K(m) values for wild-type and E224D BoNT/A LC were similar, 42 and 50 microM, respectively. Global structure, in terms of secondary structure content and topography of aromatic amino residues, Zn(2+) content, and substrate binding ability are retained in the enzymatically inactive mutants. Titration of Zn(2+) to EDTA-treated wild-type and mutant proteins indicated identical enthalpy for Zn(2+) binding. These results suggest an essential and direct role of the carboxyl group of Glu-224 in the hydrolysis of the substrate. The location of the carboxyl group at a precise position is critical for the enzymatic activity, as replacement of Glu-224 with Asp resulted in almost total loss of the activity.
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PMID:Probing the mechanistic role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain. 1069 9


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