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 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

Glucose-dependent insulinotropic polypeptide or gastric inhibitory polypeptide (GIP) is a 42 amino acid intestinal hormone, which exhibits several direct and indirect effects on fat and glucose metabolism. To determine the bioactive region(s) of the molecule, synthetic and proteolytic fragments of the hormone were generated and tested for their ability to induce a biological response in the isolated, perfused rat pancreas and stomach. A synthetic fragment corresponding to porcine GIP residues 1-30 retained strong insulinotropic activity in the isolated, perfused rat pancreas but greatly reduced somatostatinotropic activity in the isolated perfused rat stomach. A synthetic fragment corresponding to porcine GIP residues 15 to 30 was biologically inactive. However, enterokinase treatment of the synthetic 15-30 fragment restored partial insulinotropic activity in the isolated, perfused rat pancreas. The hypothesis that the restoration of biological activity was due to the enzymatic removal of the amino-terminal dipeptide (Asp-Lys) of GIP15-30 was supported by the observation that a synthetic fragment lacking these two residues was also insulinotropic. Further fractionation of the molecule generated a biologically active 19-30 fragment, suggesting that the residues necessary for the insulin response are contained within this region.
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PMID:The insulinotropic region of gastric inhibitory polypeptide; fragment analysis suggests the bioactive site lies between residues 19 and 30. 896 53

The release of cytotoxic granule contents by cytotoxic T lymphocytes triggers apoptotic target cell death. Cytotoxic granules contain a pore-forming protein, perforin, and a group of serine proteases called granzymes. We expressed human granzyme A in bacteria as a proenzyme capable of in vitro activation by enterokinase. The recombinant activated enzyme has catalytic activity against substrates with Arg, preferably, or Lys at the P1 position, comparable to trypsin. An enzymatically inactive recombinant granzyme A, with the active site Ser mutated to Ala, was produced and used with affinity chromatography to identify potential substrates. Two granzyme A-binding cytoplasmic proteins of molecular mass 33 and 44 kDa were isolated and identified by tryptic fragment sequencing as PHAP I and II, ubiquitous putative HLA-associated proteins, previously coisolated by binding to an HLA class II peptide. PHAP II forms an SDS-stable complex with recombinant mutant granzyme A and coprecipitates with it from cytoplasmic extracts. PHAP II, either purified or in cell lysates, is cleaved by the recombinant enzyme at nanomolar concentrations to a 25-kDa fragment. PHAP II begins to be degraded within minutes of initiation of cytotoxic T lymphocyte attack. PHAP I and II are candidate participants in the granzyme A pathway of cell-mediated cytotoxicity.
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PMID:Recombinant human granzyme A binds to two putative HLA-associated proteins and cleaves one of them. 925 74

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.
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PMID:Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase. 937 Mar 74

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

Enterokinase (EK) is a heterodimeric serine protease which plays a key role in initiating the proteolytic digestion cascade in the mammalian duodenum. The enzyme acts by converting trypsinogen to trypsin via a highly specific cleavage following the pentapeptide recognition sequence (Asp)4-Lys. This stringent site specificity gives EK great potential as a fusion protein cleavage reagent. Recently, a cDNA encoding the catalytic (light) chain of bovine enterokinase (EKL) was identified, characterized, and transiently expressed in mammalian COS cells. We report here the production of EKL in Escherichia coli by a novel secretory expression system that utilizes E. coli DsbA protein as an N-terminal fusion partner. The EKL cDNA was fused in-frame to the 3'-end of the coding sequence for DsbA, with the two domains of the fusion protein separated by a linker sequence encoding an enterokinase recognition site. Active, processed recombinant EKL (rEKL) was generated from this fusion protein via an autocatalytic cleavage reaction. The enzymatic properties of the bacterially produced rEKL were indistinguishable from the previously described COS-derived enzyme. Both forms of rEKL were capable of cleaving peptides, polypeptides and trypsinogen with the same specificity exhibited by the native heterodimeric enzyme purified from bovine duodena. Interestingly, rEKL activated trypsinogen poorly relative to the native heterodimeric enzyme, but was superior in its ability to cleave artificial fusion proteins containing the (Asp)4-Lys recognition sequence.
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PMID:Production of recombinant bovine enterokinase catalytic subunit in Escherichia coli using the novel secretory fusion partner DsbA. 963 75

We describe the heterologous expression of a 26.3 kD protein containing the catalytic domain of bovine enterokinase (EKL) in the methylotrophic yeast Pichia pastoris. A highly active protein is secreted and glycosylated, and it has the native amino-terminus of EKL. The cDNA encoding EKL was cloned with the KEX2 protease cleavage site following the alpha mating factor prepro secretion signal from Saccharomyces cerevisiae. The secreted EKL was easily purified from the few native proteins found in the P. pastoris fermentation supernatant, using ion exchange and affinity chromatography. The yield of the purified EKL was 6.3 mg per liter of fermentation culture. This is significantly higher than previous reports of expressions in E. coli and COS cells. The ability of this highly specific protease to cleave immediately after the carboxyl-terminal residue of the (Asp)4-Lys recognition sequence allows regeneration of native amino-terminal residues of recombinant proteins. Its application is demonstrated by the removal of thioredoxin (TrxA), and polyhistidine fusion partners from proteins of interest.
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PMID:Production of a recombinant bovine enterokinase catalytic subunit in the methylotrophic yeast Pichia pastoris. 963 16

As a step toward studying membrane fusion with a simplified molecule, the ectodomain, residues 1-185, of the membrane-anchored subunit HA2 of the influenza virus haemagglutinin (HA) was solubilized by adding the very polar FLAG octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to the N-terminal HA2 fusion peptide. The resulting chimeric protein, F185, when expressed in bacteria, folded spontaneously into a soluble trimer, with a high alpha-helical content and a high melting temperature, structural characteristics of the low-pH-induced conformation of HA2. Removal of the FLAG octapeptide by proteolysis with enterokinase converted the soluble molecule to one that aggregated, bound nonionic detergent, and bound to lipid vesicles, properties of the low-pH-induced conformation of HA. Thermolysin treatment of the aggregated protein removed the nonpolar fusion peptide, regenerating soluble trimers of HA2 (residues 24-185), which is analogous to thermolysin treatment of HA in the low-pH-induced conformation. Thermolysin treatment also dissociates F185 from the detergent-protein complex by removing the fusion peptide. These results suggest that highly polar peptides can be fused to the membrane-binding regions of membrane proteins to increase their solubility. They also indicate that ectodomains of HA2 made in bacteria have membrane-binding properties similar to those of the same ectodomain generated by low-pH treatment of HA isolated from virus.
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PMID:A polar octapeptide fused to the N-terminal fusion peptide solubilizes the influenza virus HA2 subunit ectodomain. 975 51

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

Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.
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PMID:Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide. 1049 81


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