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 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

The specificity of the synthetic substrate Gly-[L-Asp]4-L-Lys 2-naphthylamide originally developed for the assay of enteropeptidase (EC 3.4.21.9), was investigated with partially purified aminopeptidase. Our results indicate that, not only enteropeptidase, but also the concerted action of the aminopeptidases of the rat small intestine, can rapidly release 2-naphthylamine from the substrate. A previously undescribed, highly active, dipeptidylaminopeptidase, which hydrolyses a Gly-Asp dipeptide from the N-terminus of the substrate, was detected in rat small intestine. The resulting [L-Asp]3-L-Lys 2-naphthylamide fragment is then degraded by a combination of aminopeptidase A and N to yield free 2-naphthylamine. Thus the present substrate cannot be regarded as being specific for enteropeptidase, and its use leads to an over-estimation of enteropeptidase activity in homogenates and extracts of intestinal tissue. In order to prevent this non-specific hydrolysis by aminopeptidases, stereoisomeric substrates with the sequence L-Ala-D-Asp-[L-Asp]3-L-Lys methyl ester, D-Ala-[L-Asp]4-L-Lys methyl ester and L-Ala-[Asp]4-L-Lys methyl ester were synthesized and tested as alternative substrates by their ability to inhibit the enteropeptidase-catalysed activation of trypsinogen.
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PMID:Specificity studies on enteropeptidase substrates related to the N-terminus of trypsinogen. 329 38

A novel form of gastric inhibitory polypeptide (GIP), later also referred to as glucose-dependent insulinotropic polypeptide, has been isolated from bovine upper intestine. The purification was monitored by a recently developed radioreceptor assay, specific for GIP, using membrane preparations from hamster beta-cell tumors. A combination of ion-exchange and reverse-phase high-performance liquid chromatography was used in the isolation which resulted in homogeneous bovine GIP. Bovine GIP is, like porcine GIP, composed of 42 amino acid residues. The sequence is: Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-Arg- Gln-Gln - Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Ser-Asp-Trp-Ile-His- Asn-Ile - Thr-Gln, which differs from that of the previously characterized porcine GIP by having isoleucine instead of lysine at position 37. Upon proteolytic digestion of GIP with the staphylococcal V8 protease and with enterokinase, two fragments are formed in each case, corresponding to GIP1-3, GIP4-42, and GIP1-16, GIP17-42, respectively.
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PMID:A novel form of gastric inhibitory polypeptide (GIP) isolated from bovine intestine using a radioreceptor assay. Fragmentation with staphylococcal protease results in GIP1-3 and GIP4-42, fragmentation with enterokinase in GIP1-16 and GIP17-42. 639 23

The application of a new synthetic substrate to the direct determination of enteropeptidase is described. The substrate Gly-(L-Asp)4-L-Lys-2-naphthylamide contains the amino acid sequence of the activation peptides of trypsinogen linked via an amide bond to the fluorophore 2-naphthylamine. The sequence of amino acids is responsible for the specificity and substrate recognition of the enteropeptidase-catalyzed activation of trypsinogen. Interference in the assay by trypsin is prevented by the addition of soybean trypsin inhibitor to the substrate solution. The fluorimetric determination of the liberated 2-naphthylamine allows the direct observation of the reaction kinetics. For the hyrolysis of the synthetic substrate by purified enteropeptidase the pH optimum was 8.2 and the Km 0.17 mmol/l. The new substrate was used to determine the distribution of enteropeptidase along the rat small intestine and also to measure enteropeptidase activity in human intestinal biopsies.
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PMID:The application of a new synthetic substrate to the determination of enteropeptidase in rat small intestine and human intestinal biopsies. 698 11

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

The phosphorylation of human phenylalanine hydroxylase by cyclic AMP-dependent protein kinase was studied using recombinant enzyme expressed as a fusion protein in the pMAL system of Escherichia coli. Using the target sequence of the restriction protease enterokinase (Asp4-Lys) as the linker peptide, 100% full-length human phenylalanine hydroxylase was obtained on protease cleavage. The fusion protein and human phenylalanine hydroxylase were both phosphorylated at Ser-16 with a stoichiometry of 1 mol of Pi/mol of subunit. The rate of phosphorylation of human phenylalanine hydroxylase was inhibited about 40% by the cofactor tetrahydrobiopterin, and this inhibition was completely prevented by the simultaneous presence of L-phenylalanine (i.e. at turnover conditions). Phosphorylated enzyme revealed a 1.6-fold higher specific activity than the non-phosphorylated enzyme form, and it also required a lower concentration of L-Phe for substrate activation. Pre-incubation with L-Phe increased the specific activity of phenylalanine hydroxylase 2- to 4-fold, L-Phe acting with positive cooperativity. Thus, the basic catalytic and regulatory properties of recombinant human phenylalanine hydroxylase, as well as those observed for the enzyme as a fusion protein, are similar to those previously reported for the rat liver enzyme. When the target sequence of the restriction protease factor Xa (Ile-Glu-Gly-Arg) was used as the linker between maltose-binding protein and human phenylalanine hydroxylase, cleavage of the fusion protein gave a mixture of full-length hydroxylase and a truncated form of the enzyme lacking the 13 N-terminal residues. Interestingly, phosphorylation of the fusion protein, before exposure to factor Xa, almost completely protected against secondary cleavage by this restriction protease at Arg-13 of phenylalanine hydroxylase.
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PMID:Phosphorylation of recombinant human phenylalanine hydroxylase: effect on catalytic activity, substrate activation and protection against non-specific cleavage of the fusion protein by restriction protease. 857 72

The cDNA encoding a novel isoform of human trypsinogen was identified. The isoelectric points of the proenzyme and active forms calculated from the deduced amino acid sequence are consistent with those of mesotrypsin(ogen), known to be an inhibitor-resistant trypsin isoform. The cDNA attached with a bacterial signal peptide sequence was expressed in Escherichia coli. The recombinant proenzyme purified from periplasm showed enterokinase-dependent activation similar to a major isoform of human trypsinogen. The enzyme was far less inhibited by trypsin inhibitors such as soybean trypsin inhibitor, aprotinin, or pancreatic secretory trypsin inhibitor than the control trypsin. A gel filtration assay showed that the enzyme and aprotinin did not form a stable complex. It is noteworthy that the amino acid at position 198, which is in close vicinity to the active Ser, is Arg while those of other major trypsins are all Gly. It is concluded that the cloned cDNA encodes human mesotrypsinogen, a unique isoform of trypsinogen with inhibitor resistance.
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PMID:Identification and expression of the cDNA-encoding human mesotrypsin(ogen), an isoform of trypsin with inhibitor resistance. 909 3

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

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.
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PMID:Purification and further characterization of enteropeptidase from porcine duodenum. 1022 May 88


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