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)

The active site of porcine enteropeptidase (EC 3.4.21.9) was investigated in order to characterize better both catalytic and binding sites. The participation of a serine and a histidine residue in the catalytic process was fully confirmed and the two residues were located on the light chain of the enzyme. The binding site was found to be composed of at least 2 subsites S1 and S2. The subsite S1 (similar to the trypsin-binding site) is responsible for the interactions with the small substrates of trypsin and the lysine side chain of trypsinogen, while subsite S2 (probably a cluster of lysines) is responsible for the interactions with the polyanionic sequence found in all trypsinogens. Binding of substrate by subsite S2 led to an increased efficiency of the catalytic site which can be correlated to the known high specificity of enteropeptidase.
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PMID:On the catalytic and binding sites of porcine enteropeptidase. 1 10

A specific radioimmunoassay has been developed for human pancreatic cationic trypsin. The assay has been employed for the determination of immunoreactive forms of pancreatic cationic trypsin in blood. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone (TLCK) to prevent binding of the tracer to the serum inhibitors while maintaining its immunoreactivity. The average normal serum level determined was 26 ng/ml, with a range of 12--41 ng/ml. Eight of nine patients with acute pancreatic inflammation had at least a 15-fold elevation of total serum immunoreactive cationic trypsin. Cationic trypsinogen and cationic trypsin bound to alpha1-antitrypsin cross-react strongly in the radioimmunoassay. Thus it is possible to measure these potential molecular forms of cationic trypsin in serum. When normal human serum was fractionated on Sephadex G-200, all of the immunoreactive material eluted as a single peak of approximately 23,000 mol wt. No cationic trypsin could be detected in association with alpha1-antitrypsin or alpha2-macroglobulin. The 23,000-mol-wt peak was definitively shown to contain trypsinogen by affinity chromatography and by activation with human enteropeptidase. The identification of cationic trypsinogen in blood implies that the zymogen is secreted into the circulation by the pancreas rather than entering the bloodstream via absorption from the intestine.
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PMID:Determination of human pancreatic cationic trypsinogen in serum by radioimmunoassay. 43 51

The activities of highly purified human enterokinase (enteropeptidase, EC 3.4.21.9) and bovine trypsin were tested against three synthetic substrates alpha-N-Benzoyl-L-arginine ethyl ester HCl, alpha-N-Benzoyl-DL-arginine-p-nitroanilide HCl and alpha-N-Benzoyl-DL-arginine-2-naphthylamide HCl. There was no detectable hydrolysis of these substrates by enterokinase whereas the kinetic parameters obtained for trypsin were in close agreement with those previously described by other workers. The values for Km and kcat were dependent on the Ca2+ concentration. Hydrolysis of glycine-tetra-L-aspartyl-L-lysyl-2-naphthylamide (Gly(Asp)4-Lys-Nap) by these protease was also studied. Enterokinase-catalysed hydrolysis obeyed simple steady-state kinetics and values for Km of 0.525 mM and 0.28 mM and for kcat of 21.5 s-1 and 28.3 s-1 were obtained in 0.1 mM and 10 mM Ca2+, respectively. Trypsin-catalysed hydrolysis was complex and the response to Ca2+ was sigmoidal partly due to the lability of trypsin at low Ca2+ concentrations. A sensitive specific assay for enterokinase was developed and applied to the measurement of the enzyme in serum; interference by nonspecific arylamidases was eliminated by the addition of Zn2+.
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PMID:Hydrolysis of artificial substrates by enterokinase and trypsin and the development of a sensitive specific assay for enterokinase in serum. 45 24

Human cationic trypsinogen is activated by human enteropeptidase much more readily than bovine trypsinogen, the ratios kcat/Km being 330 and 11 mM-1S-1, respectively. Conversely, porcine enteropeptidase activates bovine trypsinogen much more rapidly (kcat/Km = 630 mM-1S-1) than human cationic trypsinogen (kcat/Km = 2.4 mM-1S-1). The primary structure of the activation region of human cationic trypsinogen has been investigated in an attempt to elucidate the basis for these findings. The sequence of the first 12 residues at the NH2-terminus of human cationic trypsinogen has been shown to be Asp-Lys-Ile-Val-Gly-Gly-Tyr-Asn-Cys-Glu-Glu-Asn. Furthermore, the activation peptide derived from human cationic trypsinogen has been isolated and shown to be the dipeptide Asp-Lys. This result is in contrast to the Val-(Asp)4-Lys activation peptide from bovine trypsinogen and demonstrates that human cationic trypsinogen does not contain the (Asp)4 sequence present in many other mammalian trypsinogens. It is proposed that the high degree of specificity for activation of human cationic trypsinogen by human enteropeptidase is due to the preferential recognition of the novel activation peptide sequence in the human zymogen. Thus, these two functionally related proteins, cationic trypsinogen and enteropeptidase, may have evolved in a parallel manner in the human lineage.
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PMID:Structural basis for the specific activation of human enteropeptidase. 56 6

Bovine enterokinase was purified from duodenal mucosa. The purification included an initial extraction with 2% deoxycholate, ammonium sulfate fractionations, DEAE-cellulose chromatography, and affinity chromatography on basic pancreatic trypsin inhibitor (Kunitz) (PTI)-Sepharose. The purified enzyme contained 35% carbohydrate; it had a molecular weight of 150,000, with a heavy (115,000) and light (35,000) chain connected by one or more disulfide bonds. Enterokinase hydrolyzed lysine and arginine substrates and slowly reacted with the trypsin active site titrant 4-methylumbelliferyl-p-guanidinobenzoate. The enzyme activated bovine trypsinogen with kinetic parameters similar to those of other preparations of enterokinase. Bovine enterokinase was inhibited by Kunitz pancreatic trypsin inhibitor with a Kassoc of 2 X 10(8) M-1 and only weakly by other proteinase inhibitors. The amino acid composition differed from bovine enterokinase isolated from duodenal contents (Anderson, L.E., Walsh, K.A., and Neurath, H. (1977) Biochemistry 16, 3354-3360). The mucosal enzyme and the duodenal contents enzymes also differed in the size of the heavy and light chains. The mucosal enterokinase more closely resembled the properties of porcine enterokinase (Baratti, J., Maroux, S., Louvard, D., and Desnuelle, P. (1973) Biochim. Biophys. Acta 315, 147-161). The amino acid composition and size of the light chain were also similar to bovine trypsin.
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PMID:The preparation and properties of bovine enterokinase. 76 66

The purification and characterization of three pancreatic trypsinogens A1, A2, and A3, from the African lungfish, Protopterus aethiopicus, is reported. These zymogens are activated by trypsin, by enterokinase, by an acid protease from Aspergillus oryzae, and by autoactivation. The three trypsinogens contain the same amino-terminal amino acid sequence, beginning with the activation peptide Leu-Pro-Leu-Glu-Asp-Asp-Lys-. Like the activation peptide of the previously characterized trypsinogen B [Reeck, G. R., & Neurath, H. (1972) Biochemistry 11, 503] of the same organism, it lacks the tetraaspartyl sequence characteristic of other vertebrate trypsinogens. Two of the corresponding lungfish trypsins were found to have identical amino-terminal sequences for at least 27 residues. These data suggest that the three enzymes are allelic variants. In contrast, the amino acid sequences differ sufficiently from that of trypsinogen B of the same organism to indicate that trypsinogens A and B are the products of different gene loci.
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PMID:Isolation and amino-terminal sequence analysis of a new pancreatic trypsinogen of the African lungfish Protopterus aethiopicus. 91 66

An endoproteolytic activity that specifically cleaves CCK 33, producing CCK 8, has been purified from a rat brain synaptosome preparation. The purification, which included anion exchange, chromatofocusing, hydroxyapatite, and gel filtration chromatography, resulted in a greater than 3000-fold increase in specific activity. This neutral endoprotease (pH optimum 8) exists as a 90-kDa species, which can be dissociated into active 40-kDa species. The enzyme is a non-trypsin serine protease, which is inhibited by diisopropyl-fluorophosphate and p-aminobenzamidine but not by soybean trypsin inhibitor, phenylmethylsulfonyl fluoride, aprotinin, or a number of thiol or metalloprotease inhibitors. It is highly substrate-specific and cleaves neither trypsin, enteropeptidase, kallikrein substrates, nor analogues of mono- or dibasic cleavage sites of prohormones other than pro-CCK. The endoprotease will not cleave CCK 12 desulfate or CCK (20-29), although these peptides contain common sequences with CCK-33. The protease does cleave [Glu27]CCK (20-29), a peptide in which the glutamate mimics the negative charge normally present on tyrosine sulfate. This suggests that the negative charge at position 27 is important in substrate recognition. The enzyme will also cleave CCK 33 and CCK (1-21) on the carboxyl-terminal side of a single lysine residue in position 11. The subcellular location and specificity of this endoprotease make it a good candidate for a CCK-processing protease.
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PMID:Characterization of a cholecystokinin 8-generating endoprotease purified from rat brain synaptosomes. 152 68

E. coli strains producing a hybrid protein, containing adrenocorticotropic hormone (ACTH) and protein A of S. aureus was obtained. The sequence coding for ACTH was obtained from the bovine proopiomelanocortin cDNA and, after the modification of the 5'- and 3'-terminal parts, was linked with the protein A gene and its derivatives due to synthetic adaptors. Three forms of ACTH gene, coding this hormone with differing N-terminal amino acid were used to construct the fusion gene. The hybrid proteins contain Asp-Pro or (Asp)4-Lys sequences for obtaining ACTH by acid or enterokinase treatment, respectively. It is shown that each of the constructed plasmids direct the synthesis of hybrid protein in E. coli. This protein was purified by the use of IgG-sepharose. The level of the expression of the hybrid protein is 4 mg/l of the bacterial culture. Most of the synthesized protein is secreted into the periplasmic space.
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PMID:[Expression in Escherichia coli of hybrid genes containing sequences coding the bovine adrenocorticotropic hormone]. 216 93

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

The aspartic acid residue at the bottom of the substrate-binding pocket of trypsin was replaced by glutamic acid through site-directed mutagenesis. The wild-type (Asp-189) and mutant (Glu-189) trypsinogens were expressed in E. coli, purified to homogeneity, activated by enterokinase, and tested on a series of fluorogenic tetrapeptide substrates. The substrates were of the general formula succinyl-Ala-Ala-Pro-X-AMC, where AMC is 7-amino-4-methylcoumarin and X is Lys, Arg, or Orn (ornithine). As compared to Asp-189 trypsin, the activity of Glu-189 trypsin on lysyl and arginyl substrates decreased by 3-4 orders of magnitude while its Km values did not significantly change. Lengthening the side-chain of Asp-189 by one methylene group could not be compensated for by shortening the side-chain of the substrate, since Glu-189 trypsin had no measurable activity on the ornithyl substrate. The replacement of Asp-189 with glutamic acid at the base of the substrate-binding pocket of trypsin appears to distort the structure of the critical transition-state complex. This could happen by disrupting interactions normally associated with Asp-189, and by altering the relative position of the scissile peptide bond in the active site of the enzyme.
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PMID:Structural and functional integrity of specificity and catalytic sites of trypsin. 290 52


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