Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.9 (enterokinase)
675 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine enterokinase (enteropeptidase) is a serine protease and functions as the physiological activator of trypsinogen. The enzyme has a heavy chain (115 kD) covalently linked to a light or catalytic subunit (35 kD). The amino acid composition showed that the light chain has nine half-cystine residues (four as intramolecular disulfides) and that one half-cystine was in a disulfide link between the light and heavy subunits. The amino-terminal 27 residues of the S-vinylpyridyl derivative of the light chain were determined by gas-phase Edman degradation. The sequence has homologies with other serine proteases containing one or two chains. The homologies suggest that the catalytic subunit has the same three-dimensional structure and, therefore, the same mechanism of enzymatic action as pancreatic chymotrypsin, trypsin, and elastase. The presence of the conserved amino-terminal activation peptide sequence (IVGG) shows that enterokinase must have a zymogen precursor and that the two-chain enzyme arises from limited proteolysis during posttranslational processing.
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PMID:The amino-terminal sequence of the catalytic subunit of bovine enterokinase. 179 6

The serine protease enterokinase is the physiological activator of trypsinogen and has a specificity for the sequence (Asp)4-Lys-Ile. The enzyme consists of two subunits linked by a disulfide bond. The heavy chain achors enterokinase in the intestinal brush border membrane and the light chain is the catalytic subunit, which has the same mechanism of action as trypsin and chymotrypsin. Many properties of enterokinase resemble blood-clotting enzymes, suggesting that enterokinase lies on the same phylogenetic branch as the blood-clotting proteins.
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PMID:Enterokinase (enteropeptidase): comparative aspects. 265 18

Enterokinase is a serine protease of the duodenal brush border membrane that cleaves trypsinogen and produces active trypsin, thereby leading to the activation of many pancreatic digestive enzymes. Overlapping cDNA clones that encode the complete human enterokinase amino acid sequence were isolated from a human intestine cDNA library. Starting from the first ATG codon, the composite 3696 nt cDNA sequence contains an open reading frame of 3057 nt that encodes a 784 amino acid heavy chain followed by a 235 amino acid light chain; the two chains are linked by at least one disulfide bond. The heavy chain contains a potential N-terminal myristoylation site, a potential signal anchor sequence near the amino terminus, and six structural motifs that are found in otherwise unrelated proteins. These domains resemble motifs of the LDL receptor (two copies), complement component Clr (two copies), the metalloprotease meprin (one copy), and the macrophage scavenger receptor (one copy). The enterokinase light chain is homologous to the trypsin-like serine proteinases. These structural features are conserved among human, bovine, and porcine enterokinase. By Northern blotting, a 4.4 kb enterokinase mRNA was detected only in small intestine. The enterokinase gene was localized to human chromosome 21q21 by fluorescence in situ hybridization.
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PMID:cDNA sequence and chromosomal localization of human enterokinase, the proteolytic activator of trypsinogen. 771 57

Enteropeptidase (EC 3.4.21.9) is a key enzyme in the intestinal digestion cascade responsible for the conversion of trypsinogen to trypsin, which then activates various pancreatic zymogens. In order to structurally characterize the enzyme, we purified the enzyme from porcine duodenal mucosa and showed that it consists of three polypeptide chains, which we named "mini" chain (M chain), light chain (L chain), and heavy chain (H chain) in order of increasing molecular size. Based on their NH2-terminal sequences, a cDNA clone for porcine enteropeptidase was isolated and analyzed. The clone was 3597 base pairs long, which encoded 1034 amino acid residues of a single-chain precursor form of enteropeptidase. The precursor contained an additional NH2-terminal 51-residue sequence including a putative internal signal sequence, followed by the M chain (66 residues), the H chain (682 residues), and the L chain (235 residues) in that order. The H chain had regions partially homologous in sequence with low density lipoprotein receptor and complement components. On the other hand, the L chain was highly homologous with the catalytic domains of trypsin-like serine proteinases. The structural model of the L chain suggests that the sequence, Arg885-Arg-Arg-Lys888, is probably involved in the unique substrate specificity of the enzyme, preferring acidic amino acid residues at the P2-P5 sites.
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PMID:Structural characterization of porcine enteropeptidase. 805 Oct 81

Enterokinase is a protease of the intestinal brush border that specifically cleaves the acidic propeptide from trypsinogen to yield active trypsin. This cleavage initiates a cascade of proteolytic reactions leading to the activation of many pancreatic zymogens. The full-length cDNA sequence for bovine enterokinase and partial cDNA sequence for human enterokinase were determined. The deduced amino acid sequences indicate that active two-chain enterokinase is derived from a single-chain precursor. Membrane association may be mediated by a potential signal-anchor sequence near the amino terminus. The amino terminus of bovine enterokinase also meets the known sequence requirements for protein N-myristoylation. The amino-terminal heavy chain contains domains that are homologous to segments of the low density lipoprotein receptor, complement components C1r and C1s, the macrophage scavenger receptor, and a recently described motif shared by the metalloprotease meprin and the Xenopus A5 neuronal recognition protein. The carboxyl-terminal light chain is homologous to the trypsin-like serine proteases. Thus, enterokinase is a mosaic protein with a complex evolutionary history. The amino acid sequence surrounding the amino terminus of the enterokinase light chain is ITPK-IVGG (human) or VSPK-IVGG (bovine), suggesting that single-chain enterokinase is activated by an unidentified trypsin-like protease that cleaves the indicated Lys-Ile bond. Therefore, enterokinase may not be the "first" enzyme of the intestinal digestive hydrolase cascade. The specificity of enterokinase for the DDDDK-I sequence of trypsinogen may be explained by complementary basic-amino acid residues clustered in potential S2-S5 subsites.
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PMID:Enterokinase, the initiator of intestinal digestion, is a mosaic protease composed of a distinctive assortment of domains. 805 24

Enterokinase (enteropeptidase) is a heterodimeric serine protease that is responsible for the physiological activation of trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)4-Lys. In this paper, we report the cloning and functional expression of a cDNA encoding the catalytic domain (light chain) of bovine enterokinase. The nucleotide sequence of this cloned cDNA predicts a 235-amino acid polypeptide that shares a high degree of homology with a variety of mammalian serine proteases involved in digestion, coagulation, and fibrinolysis. We have developed a novel expression method for the enzyme which utilizes the secretory leader and propeptide of the mammalian serine protease PACE fused to the enterokinase light chain amino terminus. Efficient cleavage of the paired dibasic amino acid cleaving enzyme (PACE) propeptide was achieved by coexpression with human PACE or yeast KEX2. The mature product migrates at 43,000 Da on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, comparable to light chain derived from bovine duodena, and exhibited high levels of activity in cleaving the enterokinase-specific fluorogenic substrate Gly-(Asp)4-Lys-beta-naphthylamide. The recombinant single-chain form of enterokinase was also capable of activating trypsinogen, indicating that the specificity of the enzyme for its natural substrate is retained even in the absence of the noncatalytic enterokinase heavy chain.
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PMID:Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. 822 55

Enteropeptidase, also known as enterokinase, initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. Enteropeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximately 47-kDa serine protease domain (light chain) and a disulfide-linked approximately 120-kDa heavy chain. The heavy chain contains an amino-terminal membrane-spanning segment and several repeated structural motifs of unknown function. To study the role of heavy chain motifs in substrate recognition, secreted variants of recombinant bovine proenteropeptidase were constructed by replacing the transmembrane domain with a signal peptide. Secreted variants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enteropeptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified. Single-chain pro-HL-BEK and pro-L-BEK were zymogens with extremely low catalytic activity, and both were activated readily by trypsin cleavage. Trypsinogen was activated efficiently by purified enteropeptidase from bovine intestine (Km = 5.6 microM and kcat = 4.0 s-1) and by HL-BEK (Km = 5.6 microM and kcat = 2.2 s-1), but not by L-BEK (Km = 133 microM and kcat = 0.1 s-1); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater catalytic efficiency than did L-BEK. Qualitatively similar results were obtained at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (Km = 0.27 mM and kcat = 0.07 s-1) and L-BEK (Km = 0.60 mM and kcat = 0.06 s-1). The presence of the heavy chain also influenced the rate of reaction with protease inhibitors. Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial Ki = 99 nM and final Ki* = 1.8 nM) over L-BEK (Ki = 698 nM and Ki* = 6.2 nM). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (Ki* = 1.6 nM), but not HL-BEK. These kinetic data indicate that the enteropeptidase heavy chain has little influence on the recognition of small peptides, but strongly influences macromolecular substrate recognition and inhibitor specificity.
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PMID:Bovine proenteropeptidase is activated by trypsin, and the specificity of enteropeptidase depends on the heavy chain. 939 56

The effects of structural modification upon the specificity of enteropeptidase were studied. A variation in the unique specificity of the enzyme was shown to be the result of an autolysis caused by the enzyme's loss of calcium ions. The cleavage sites of the autolysis were determined. A truncated enzyme containing the C-terminal fragment of its heavy chain (466-800 residues) and the intact light chain were shown to be the products of autolysis. The kinetic parameters of the hydrolysis of trypsinogen, a recombinant protein, and a peptide substrate with both forms of enteropeptidase were determined. Conditions were found that can help regulate the transition of the native enzyme into the truncated form. A hypothesis was proposed concerning the autoactivational character of proenteropeptidase processing.
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PMID:[Structural characteristics providing for high specificity of enteropeptidase]. 961 70

Enteropeptidase is a heterodimeric type II membrane protein of the brush border of duodenal enterocytes. In this location, enteropeptidase cleaves and activates trypsinogen, thereby initiating the activation of other intestinal digestive enzymes. Recombinant bovine enteropeptidase was sorted directly to the apical surface of polarized Madin-Darby canine kidney cells. Replacement of the cytoplasmic and signal anchor domains with a cleavable signal peptide (mutant proenteropeptidase lacking the amino-terminal signal anchor domain (dSA-BEK)) caused apical secretion. The additional amino-terminal deletion of a mucin-like domain (HL-BEK) resulted in secretion both apically and basolaterally. Further deletion of the noncatalytic heavy chain (L-BEK) resulted in apical secretion. Thus enteropeptidase appears to have at least three distinct sorting signals as follows: the light chain (L-BEK) directs apical sorting, addition of most of the heavy chain (HL-BEK) inhibits apical sorting, and addition of the mucin-like domain (dSA-BEK) restores apical sorting. Inhibition of N-linked glycosylation with tunicamycin or disruption of microtubules with colchicine caused L-BEK to be secreted equally into apical and basolateral compartments, whereas brefeldin A caused basolateral secretion of L-BEK. Full-length BEK was not found in detergent-resistant raft domains of Madin-Darby canine kidney cells or baby hamster kidney cells. These results suggest apical sorting of enteropeptidase depends on N-linked glycosylation of the serine protease domain and an amino-terminal segment that includes an O-glycosylated mucin-like domain and three potential N-glycosylation sites. In contrast to many apically targeted proteins, enteropeptidase does not form detergent-resistant associations with sphingolipid-cholesterol rafts.
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PMID:Apical sorting of bovine enteropeptidase does not involve detergent-resistant association with sphingolipid-cholesterol rafts. 988 May 38

Variations in bovine enteropeptidase (EP) activity were shown to result from autolysis caused by the loss of calcium ions; the cleavage sites were determined. The native enzyme preferred its natural substrate, trypsinogen (KM=2.4 microM), to the peptide and fusion protein substrates (KM=200 and 125 microM, respectively). On the other hand, the truncated enzyme composed of the C-terminal fragment 466-800 of EP heavy chain and intact light chain did not distinguish these substrates. The results suggest that the N-terminal fragment 118-465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.
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PMID:Autolysis of bovine enteropeptidase heavy chain: evidence of fragment 118-465 involvement in trypsinogen activation. 992 6


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