Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.11 (CD10)
 9,792 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

After enzymatic digestion of chicken myoglobin by trypsin, chymotrypsin or thermolysin, the separation of peptides was performed by column chromatography on various ion exchange resins. Each peptide was purified by high-voltage paper electrophoresis or by chromatography either on paper or on ion-exchange resin, and its complete amino acid sequence was then determined by the combined dansyl-Edman procedure and by endopeptidase digestions. The whole globin was submitted to automatic Edman degradation using the Beckman sequencer. Residues have been positioned from overlaps of sequence data between tryptic (T), chymotryptic (C) and thermolysin (Th) peptides. The stepwise degradation of the whole globin confirmed the alignment of the N-terminal third of the molecule. The combination of these different approaches has led to the complete determination of the 153 residues sequence forming the polypeptide chain of chicken myoglobin. Comparison of the established chicken myoglobin structure with those from other species shows a conservation of structure, although the avian protein exhibits more variations in its amino acid sequence than has been found between other known myoglobins which all belong to mammalian species.
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PMID:The primary sequence of chicken myoglobin (Gallus gallus). 116 72

The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var. amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.
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PMID:Degradation of streptomyces metalloprotease inhibitor (SMPI) by neutral protease from Bacillus subtilis var. amylosacchariticus. 136 40

To further analyze CD10/NEP function in lymphoid and nonlymphoid cells using well characterized murine systems, we isolated the murine CD10/NEP homologue, determined its chromosomal location, and modeled the enzyme's active site. The murine CD10/NEP cDNA predicts a 750-amino acid (aa) type II integral membrane protein with 90% identity to the human CD10 sequence and 100% conservation of critical aa and functional motifs. The latter include the pentapeptide consensus sequence required for zinc binding and catalytic activity, additional aa associated with substrate binding, and the extracellular cysteines that participate in disulfide bonds required for enzymatic activity. Like its human homologue, murine CD10/NEP has multiple alternative 5'-untranslated region sequences. The gene is localized on the proximal half of murine chromosome 3. In Northern analysis, murine CD10/NEP transcripts are abundant in bone marrow stromal cells that support pre-B cell differentiation but are undetectable in representative Abelson transformed pre-B cell lines. The murine CD10/NEP active site was modeled by aligning critical conserved CD10/NEP residues with comparable residues in the active site of thermolysin, a bacterial metalloprotease with similar substrate specificity. The model predicts that the two enzymes have similar clefts that comprise the active site and permit zinc-dependent substrate interactions.
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PMID:Murine common acute lymphoblastic leukemia antigen (CD10 neutral endopeptidase 24.11). Molecular characterization, chromosomal localization, and modeling of the active site. 137 1

A novel metallo-endopeptidase from human neuroblastoma NB-OK-1 cells was partially purified and characterized. This enzyme activity was detected in the culture medium and could be detached from intact cells by gentle washing, suggesting a peripheral localization of the enzyme. This endopeptidase inactivated Atrial Natriuretic Peptide (ANP) by a unique and selective cleavage of the Ser123-Phe124 bond. It also produced hydrolysis at the Xaa-Phe, Xaa-Leu, or Xaa-Ile bonds of other peptide hormones such as bradykinin, somatostatin 14, litorin, substance P, neuromedin C and angiotensin II. The substrate selectivity and inhibition profile of the enzyme showed obvious similarities with the peptide hormone inactivating endopeptidase (PHIE) recently purified from Xenopus laevis skin secretions and indicated a thermolysin-like activity distinct from neutral endopeptidase (EC 3.4.24.11) and from angiotensin converting enzyme (EC 3.4.15.1).
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PMID:A new metallo- endopeptidase from human neuroblastoma NB-OK-1 cells which inactivates atrial natriuretic peptide by selective cleavage at the Ser123-Phe124 bond. 153 Oct 11

Sequence analysis of aminopeptidase N has shown that this zinc exopeptidase contains a consensus sequence (Val-Xaa-Xaa-His-Glu-Xaa-Xaa-His), generally found at the active site of zinc endopeptidases [Jongeneel, C. V., Bouvier, J. and Bairoch, A. (1989) FEBS Lett. 242, 211-214]. This suggests that the active site of aminopeptidase N may be closer to that of a classical zinc endopeptidase, such as thermolysin, than to that of an exopeptidase, such as carboxypeptidase A, which does not contain the above sequence. However, the nature of the other amino acids involved in the enzymatic activity of the eukaryotic aminopeptidase N remains unknown. Chemical modifying agents have now been used to characterize the active site of aminopeptidase N further. The location of the modified residues was also determined by comparing the protection given by three competitive inhibitors which interact with different subsites of the active site. Aminopeptidase N was rapidly inactivated by 2,3-butanedione and diethylpyrocarbonate and partially inactivated by N-acetylimidazole, diazoacetamide and a soluble carbodiimide, suggesting the presence of functional arginyl, histidyl, tyrosyl and aspartyl/glutamyl residues. In each case the reaction kinetics showed that the inactivation could be correlated with modification of a single residue. The protection experiments indicated that the residues are at the active site of the enzyme and that the arginine and tyrosine are probably located in the S'1-S'2 subsites, histidine in the S1 subsite and the acidic residue near the zinc binding site and the S'1 subsite. Steady-state kinetics showed that the arginine, histidine and acidic residues are involved in substrate binding, while the tyrosine may play a role in the catalytic process. All these data support an endopeptidase-like structure for the active site of aminopeptidase N.
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PMID:Functional residues at the active site of aminopeptidase N. 167 19

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

The activation or interruption of the responses induced by regulatory peptides are ensured by ectoenzymes, the most important of them belonging to the group of zinc metallopeptidases. Thus angiotensin converting enzyme (ACE) forms the hypertensive peptide angiotensin II from its inactive precursor AI. This also the case for aminopeptidase N (APN) and neutral endopeptidase 24.11 (NEP, CALLA) which together inactivate the endogenous opioid peptides, enkephalins, whereas only NEP is involved in the metabolism of the atrial natriuretic factor (ANP) at the kidney and vascular levels. The pharmacological effects resulting from the inhibition of these enzymatic processes will appear only in tissues where the peptide substrate is tonically or phasically released. This promising approach is expected to avoid, or at least to minimize, the side effects resulting from excessive and ubiquitous stimulation of peptide receptors by exogenously administered agonists or antagonists. The essential amino acids known to be present in the active site of the bacterial endopeptidase thermolysin from crystallographic studies, have also been found in NEP by using a new program of sequence comparison associated with mutagenesis experiments. Several classes of selective inhibitors of NEP, APN and ACE have been rationally designed by taking into account the structural differences in the active site of these peptidases. Thus, the retro-inversion of the amide bond of the NEP inhibitor thiorphan resulted in the elimination of a residual interaction with ACE. Moreover, we have proposed to associate inhibitory potencies towards two peptidases in the same compound. Thus kelatorphan HONH-CO-CH2-CH(CH2 phi)-CONH-CH(CH3)-COOH and other systemically-active mixed NEP/APN inhibitors were shown capable of completely blocking enkephalin metabolism in vivo. This concept has been extended to mixed NEP/ACE inhibitors with compounds such as HS-CH2-CH(CH2 phi)-CONH-CH(CH2R)-COOH where R = CH-(CH3)2 (ES 34) or -OCH2 phi (ES 37). Only mixed inhibitors of NEP and APN are able to produce potent analgesia after intracerebroventricular or systemic administration without the major side effects of morphine (tolerance and dependence). Thiorphan or its prodrugs acetorphan or sinorphan lead to a increase in natriuresis and diuresis by protection of ANP degradation, but without any significant antihypertensive effect. Contrastingly mixed NEP/ACE inhibitors such as ES34 induce decreases in blood pressure higher than those that produced by the association of selective NEP and ACE inhibitors.
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PMID:[New approach in the research of analgesics and antihypertensive agents]. 184 70

Phe5(4-nitro)-bradykinin has been identified as a good synthetic substrate to study the kinetics and mechanism of action of the metalloendopeptidase meprin. No convenient substrate for kinetic analysis of the enzyme had been previously described. HPLC analyses indicated that meprin cleaved bradykinin and nitrobradykinin between Phe5 (or Phe5(NO2)) and Ser6. Reaction rates for bradykinin were determined by quantitative HPLC analyses, whereas rates for nitrobradykinin were measured by continuous monitoring of the spectral change that occurs at 310 nm when the Phe(NO2)-Ser bond is hydrolyzed. For nitrobradykinin and unmodified bradykinin, respectively, Km values were 281 and 425 microM, kcat values were 28 and 22 s-1, and kcat/Km values were 9.7 x 10(4) and 5.1 x 10(4)M-1. The two products of bradykinin hydrolysis were not substrates for the enzyme, but they were inhibitors. The initial rates of hydrolysis of nitrobradykinin increased linearly with enzyme concentration (0.09-2.2 micrograms/ml), and increased linearly with temperature in the range from 15 to 55 degrees C. Hydrolysis of the substrate was optimal at alkaline pH values. The cysteine endopeptidases papain and cathepsin L and the metalloproteases thermolysin, angiotensin-converting enzyme, and neutral endopeptidase (EC 3.4.24.11) also cleaved nitrobradykinin, but at different peptide bonds than meprin. The single cleavage of nitrobradykinin at the Phe(NO2)-Ser bond and the concomitant spectral shift that occurs at alkaline pH makes this a particularly suitable substrate for meprin.
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PMID:Phe5(4-nitro)-bradykinin: a chromogenic substrate for assay and kinetics of the metalloendopeptidase meprin. 196 66

On the basis of the identity of a segment of the amino acid sequence within the active site of the bacterial enzyme thermolysin and the mammalian enzyme neutral endopeptidase 24.11, the possible involvement of valine-573 of neutral endopeptidase 24.11 in substrate binding was investigated. Valine-573 was changed to leucine and to alanine by site-directed mutagenesis. The effect of these mutations on inhibitor binding and substrate catalysis was examined with a series of compounds containing variable P'1 residues. With a small P'1 residue such as alanine, both mutant enzymes exhibited kinetic properties essentially the same as the wild-type enzyme. However, with larger P'1 residues such as phenylalanine, tyrosine, and leucine, the Val573Leu mutant showed a 24-100-fold decrease in inhibitor affinity. Similarly substrates containing bulky P'1 residues showed a 10-40-fold decrease in Vmax with little change in Km. In contrast, the Val573Ala mutant showed only modest changes in terms of inhibitor binding or substrate turnover. These results support the proposed role of valine-573 as a part of the hydrophobic binding pocket, S'1 binding subsite, of neutral endopeptidase 24.11.
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PMID:Use of site-directed mutagenesis to identify valine-573 in the S'1 binding site of rat neutral endopeptidase 24.11 (enkephalinase). 226 63


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