Bovine Proenteropeptidase Is Activated by Trypsin, and the Specificity of Enteropeptidase Depends on the Heavy Chain

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 ≈47-kDa serine protease domain (light chain) and a disulfide-linked ≈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 (K m = 5.6 μm and k cat = 4.0 s−1) and by HL-BEK (K m = 5.6 μm and k cat = 2.2 s−1), but not by L-BEK (K m = 133 μm and k cat = 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-β-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (K m = 0.27 mm and k cat = 0.07 s−1) and L-BEK (K m = 0.60 mm and k cat = 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 K i = 99 nm and final K i * = 1.8 nm) over L-BEK (K i = 698 nm andK i * = 6.2 nm). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (K i * = 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. 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 ≈47-kDa serine protease domain (light chain) and a disulfide-linked ≈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 (K m = 5.6 μm and k cat = 4.0 s−1) and by HL-BEK (K m = 5.6 μm and k cat = 2.2 s−1), but not by L-BEK (K m = 133 μm and k cat = 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-β-naphthylamide was cleaved with similar kinetic parameters by both HL-BEK (K m = 0.27 mm and k cat = 0.07 s−1) and L-BEK (K m = 0.60 mm and k cat = 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 K i = 99 nm and final K i * = 1.8 nm) over L-BEK (K i = 698 nm andK i * = 6.2 nm). Soybean trypsin inhibitor exhibited a reciprocal pattern, inhibiting L-BEK (K i * = 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. Enteropeptidase, originally named enterokinase when it was discovered by Pavlov (1Pavlov I.P. The Work of the Digestive Glands. Charles Griffin & Co., London1902: 148-163Google Scholar), is a membrane-bound serine protease of the duodenal mucosa that cleaves trypsinogen to generate active trypsin. In almost all vertebrate species, a short trypsinogen activation peptide is released that terminates with the sequence Asp-Asp-Asp-Asp-Lys (2Bricteux-Gregoire S. Schyns R. Florkin M. Comp. Biochem. Physiol. B Comp. Biochem. 1972; 42: 23-39Crossref PubMed Scopus (35) Google Scholar). Following activation, trypsin cleaves and activates other zymogens in pancreatic secretions, including chymotrypsinogen, proelastase, procarboxypeptidases, and some prolipases. Thus, enteropeptidase initiates a simple two-step enzymatic cascade that activates digestive hydrolases within the lumen of the gut. The biological importance of this pathway is demonstrated by the severe intestinal malabsorption and diarrhea that is caused by congenital enteropeptidase deficiency (3Hadorn B. Tarlow M.J. Lloyd J.K. Wolff O.H. Lancet. 1969; i: 812-813Abstract Google Scholar, 4Haworth J.C. Gourley B. Hadorn B. Sumida C. J. Pediatr. 1971; 78: 481-490Abstract Full Text PDF PubMed Scopus (51) Google Scholar).Bovine enteropeptidase is synthesized as a single-chain precursor of 1035 amino acid residues (5Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (145) Google Scholar) that appears to require proteolytic activation, suggesting that enteropeptidase may not be the “first” protease of the digestive hydrolase cascade. Active enteropeptidase has been cleaved after Arg-800 to produce a disulfide-linked heterodimer with an amino-terminal ≈120-kDa heavy chain and a ≈47-kDa light chain; ∼40% of the apparent mass of these polypeptides is due to glycosylation (6Anderson L.E. Walsh K.A. Neurath H. Biochemistry. 1977; 16: 3354-3360Crossref PubMed Scopus (44) Google Scholar, 7Liepnieks J.J. Light A. J. Biol. Chem. 1979; 254: 1677-1683Abstract Full Text PDF PubMed Google Scholar). The enteropeptidase heavy chain consists of an amino-terminal membrane-spanning domain, a mucin-like domain, two repeats found in complement serine proteases C1r and C1s, a MAM domain (so named for similar motifs in the the and and a in Sadler J.E. of Scholar). The light chain is a serine The activation the heavy and light has the sequence be by trypsin or other The of the proenteropeptidase is trypsin were in this the of a be in Sadler J.E. of of enteropeptidase substrate not and may not be to the catalytic serine protease The enteropeptidase light chain has been in active by and of purified bovine intestinal enteropeptidase A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), with an of similar has been by of recombinant enteropeptidase light chain A. C. Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), is to at These light chain to but in trypsinogen been by (6Anderson L.E. Walsh K.A. Neurath H. Biochemistry. 1977; 16: 3354-3360Crossref PubMed Scopus (44) Google Scholar, PubMed Scopus Google Scholar) or J. S. PubMed Scopus Google Scholar) of the structural of the trypsinogen activation is these that the catalytic and at on enteropeptidase to the influence of the enteropeptidase heavy chain on substrate recognition, secreted variants of recombinant proenteropeptidase were with or the heavy chain. was to be a with extremely low protease activity, and it was activated efficiently by trypsin. The presence of the heavy chain proenteropeptidase activation and almost on the recognition of the small peptide substrate the heavy chain on the recognition of macromolecular and trypsin inhibitor and trypsin activation of trypsinogen is a in the activation of other digestive hydrolases within the lumen of the and of this reaction on all vertebrate activated by proteolytic of a in an amino-terminal peptide that contains the sequence (2Bricteux-Gregoire S. Schyns R. Florkin M. Comp. Biochem. Physiol. B Comp. Biochem. 1972; 42: 23-39Crossref PubMed Scopus (35) Google Scholar). of enteropeptidase that residues on the of the catalytic (light chain) with the residues of trypsinogen activation M. M. S. H. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). may for the recognition of small peptide but not to the recognition of trypsinogen. The light chain has been by of purified bovine enteropeptidase A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or by of recombinant light chain A. C. Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). small synthetic peptides, but trypsinogen. the recognition of small only the light whereas of trypsinogen may also on the heavy chain. in trypsinogen recognition were in enteropeptidase by (6Anderson L.E. Walsh K.A. Neurath H. Biochemistry. 1977; 16: 3354-3360Crossref PubMed Scopus (44) Google Scholar, PubMed Scopus Google Scholar) or by J. S. PubMed Scopus Google Scholar). that the catalytic and at to and to these be on the heavy chain or the light the of enteropeptidase substrate variants were constructed that or of the heavy chain. of the transmembrane segment appears to little on trypsinogen activation by bovine A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or M. M. S. H. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) the membrane-spanning domain was with a signal peptide to of secreted recombinant from was to the of the that serine proteases an that the activation peptide to the protease domain, and of enteropeptidase with a of serine proteases that the of the heavy chain is to the light chain or in (5Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (145) Google Scholar). To this and to the of an or the the amino of the heavy chain that to the activation peptide. of the short activation peptide and the catalytic domain was by that pro-L-BEK and L-BEK similar whereas after the apparent mass of L-BEK is than that of pro-L-BEK pro-HL-BEK and pro-L-BEK were purified as single-chain and were found to little catalytic In pro-HL-BEK than the of HL-BEK The activation of proenteropeptidase in the sequence has the of a for by trypsin (5Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (145) Google Scholar). In trypsin readily activated both pro-HL-BEK and pro-L-BEK by cleaving after was activated than that the heavy chain has a on the recognition of proenteropeptidase by of proenteropeptidase to were not to of the K m for by be μm not trypsin cleaved pro-HL-BEK at a rate of and pro-L-BEK at a rate of at nm), and these similar to of other trypsin Thus, proenteropeptidase is as a single-chain and it is activated efficiently by in activates the of a be in study is to trypsinogen or proenteropeptidase is for this or protease may be in duodenal mucosa or pancreatic of the heavy chain only a on proenteropeptidase activation, it a and on enteropeptidase substrate specificity. and L-BEK similar kinetic parameters for of that the heavy chain not strongly influence the recognition of small the substrate BEK and HL-BEK also similar m and the cat that the transmembrane and little on trypsinogen is with the that proteolytic of enteropeptidase from did not trypsinogen Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In contrast to these small of the heavy chain the of trypsinogen with the catalytic efficiency m of L-BEK was at pH 5.6 and at pH 8.4. heavy chain motifs the low domain and the domain strongly influence the recognition of trypsinogen. The in trypsinogen activation is not by in the of L-BEK is These results the enteropeptidase heavy chain contains an that with to trypsinogen activation, the heavy chain also the of enteropeptidase with macromolecular protease inhibitors. BEK purified from intestine was to be by with a of nm J.J. Light A. J. Biol. Chem. 1979; 254: 1677-1683Abstract Full Text PDF PubMed Google Scholar), and recombinant HL-BEK also was found to be by with a i * of 1.8 nm In the were of was with a in a low was with a K i of 99 and this to the final with a rate of of the heavy chain little on k the final K i * to 6.2 and the K i to nm was for BEK is to J.J. Light A. J. Biol. Chem. 1979; 254: 1677-1683Abstract Full Text PDF PubMed Google Scholar), and HL-BEK also was not by of the heavy chain L-BEK to by with a K i * of 1.6 nm In the of the L-BEK reaction with did not indicate the of an low enteropeptidase light chain by and A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and a recombinant with an A. C. Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) also by Thus, of the heavy chain the of enteropeptidase by but by enteropeptidase and not amino to the that with the catalytic and for this the enteropeptidase heavy chain may be a of reaction with these and other kinetic of the enteropeptidase heavy chain it in the recognition of macromolecular and inhibitors. The activation of trypsinogen is by the heavy and influence is acid that in the The of enteropeptidase to protease also appears to in on the heavy chain. The of the to of for the of serine proteases to macromolecular These serine proteases and to by structural of both catalytic and Enteropeptidase, originally named enterokinase when it was discovered by Pavlov (1Pavlov I.P. The Work of the Digestive Glands. Charles Griffin & Co., London1902: 148-163Google Scholar), is a membrane-bound serine protease of the duodenal mucosa that cleaves trypsinogen to generate active trypsin. In almost all vertebrate species, a short trypsinogen activation peptide is released that terminates with the sequence Asp-Asp-Asp-Asp-Lys (2Bricteux-Gregoire S. Schyns R. Florkin M. Comp. Biochem. Physiol. B Comp. Biochem. 1972; 42: 23-39Crossref PubMed Scopus (35) Google Scholar). Following activation, trypsin cleaves and activates other zymogens in pancreatic secretions, including chymotrypsinogen, proelastase, procarboxypeptidases, and some prolipases. Thus, enteropeptidase initiates a simple two-step enzymatic cascade that activates digestive hydrolases within the lumen of the gut. The biological importance of this pathway is demonstrated by the severe intestinal malabsorption and diarrhea that is caused by congenital enteropeptidase deficiency (3Hadorn B. Tarlow M.J. Lloyd J.K. Wolff O.H. Lancet. 1969; i: 812-813Abstract Google Scholar, 4Haworth J.C. Gourley B. Hadorn B. Sumida C. J. Pediatr. 1971; 78: 481-490Abstract Full Text PDF PubMed Scopus (51) Google Scholar). Bovine enteropeptidase is synthesized as a single-chain precursor of 1035 amino acid residues (5Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (145) Google Scholar) that appears to require proteolytic activation, suggesting that enteropeptidase may not be the “first” protease of the digestive hydrolase cascade. Active enteropeptidase has been cleaved after Arg-800 to produce a disulfide-linked heterodimer with an amino-terminal ≈120-kDa heavy chain and a ≈47-kDa light chain; ∼40% of the apparent mass of these polypeptides is due to glycosylation (6Anderson L.E. Walsh K.A. Neurath H. Biochemistry. 1977; 16: 3354-3360Crossref PubMed Scopus (44) Google Scholar, 7Liepnieks J.J. Light A. J. Biol. Chem. 1979; 254: 1677-1683Abstract Full Text PDF PubMed Google Scholar). The enteropeptidase heavy chain consists of an amino-terminal membrane-spanning domain, a mucin-like domain, two repeats found in complement serine proteases C1r and C1s, a MAM domain (so named for similar motifs in the the and and a in Sadler J.E. of Scholar). The light chain is a serine The activation the heavy and light has the sequence be by trypsin or other The of the proenteropeptidase is trypsin were in this the of a be in Sadler J.E. of Scholar). The of enteropeptidase substrate not and may not be to the catalytic serine protease The enteropeptidase light chain has been in active by and of purified bovine intestinal enteropeptidase A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), with an of similar has been by of recombinant enteropeptidase light chain A. C. Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), is to at These light chain to but in trypsinogen been by (6Anderson L.E. Walsh K.A. Neurath H. Biochemistry. 1977; 16: 3354-3360Crossref PubMed Scopus (44) Google Scholar, PubMed Scopus Google Scholar) or J. S. PubMed Scopus Google Scholar) of the structural of the trypsinogen activation is these that the catalytic and at on enteropeptidase to trypsinogen. To the influence of the enteropeptidase heavy chain on substrate recognition, secreted variants of recombinant proenteropeptidase were with or the heavy chain. was to be a with extremely low protease activity, and it was activated efficiently by trypsin. The presence of the heavy chain proenteropeptidase activation and almost on the recognition of the small peptide substrate the heavy chain on the recognition of macromolecular and trypsin inhibitor and trypsin activation of trypsinogen is a in the activation of other digestive hydrolases within the lumen of the and of this reaction on all vertebrate activated by proteolytic of a in an amino-terminal peptide that contains the sequence (2Bricteux-Gregoire S. Schyns R. Florkin M. Comp. Biochem. Physiol. B Comp. Biochem. 1972; 42: 23-39Crossref PubMed Scopus (35) Google Scholar). of enteropeptidase that residues on the of the catalytic (light chain) with the residues of trypsinogen activation M. M. S. H. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). may for the recognition of small peptide but not to the recognition of trypsinogen. The light chain has been by of purified bovine enteropeptidase A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or by of recombinant light chain A. C. Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). small synthetic peptides, but trypsinogen. the recognition of small only the light whereas of trypsinogen may also on the heavy chain. in trypsinogen recognition were in enteropeptidase by (6Anderson L.E. Walsh K.A. Neurath H. Biochemistry. 1977; 16: 3354-3360Crossref PubMed Scopus (44) Google Scholar, PubMed Scopus Google Scholar) or by J. S. PubMed Scopus Google Scholar). that the catalytic and at to and to these be on the heavy chain or the light the of enteropeptidase substrate variants were constructed that or of the heavy chain. of the transmembrane segment appears to little on trypsinogen activation by bovine A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or M. M. S. H. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) the membrane-spanning domain was with a signal peptide to of secreted recombinant from was to the of the that serine proteases an that the activation peptide to the protease domain, and of enteropeptidase with a of serine proteases that the of the heavy chain is to the light chain or in (5Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (145) Google Scholar). To this and to the of an or the the amino of the heavy chain that to the activation peptide. of the short activation peptide and the catalytic domain was by that pro-L-BEK and L-BEK similar whereas after the apparent mass of L-BEK is than that of pro-L-BEK pro-HL-BEK and pro-L-BEK were purified as single-chain and were found to little catalytic In pro-HL-BEK than the of HL-BEK The activation of proenteropeptidase in the sequence has the of a for by trypsin (5Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (145) Google Scholar). In trypsin readily activated both pro-HL-BEK and pro-L-BEK by cleaving after was activated than that the heavy chain has a on the recognition of proenteropeptidase by of proenteropeptidase to were not to of the K m for by be μm not trypsin cleaved pro-HL-BEK at a rate of and pro-L-BEK at a rate of at nm), and these similar to of other trypsin Thus, proenteropeptidase is as a single-chain and it is activated efficiently by in activates the of a be in study is to trypsinogen or proenteropeptidase is for this or protease may be in duodenal mucosa or pancreatic of the heavy chain only a on proenteropeptidase activation, it a and on enteropeptidase substrate specificity. and L-BEK similar kinetic parameters for of that the heavy chain not strongly influence the recognition of small the substrate BEK and HL-BEK also similar m and the cat that the transmembrane and little on trypsinogen is with the that proteolytic of enteropeptidase from did not trypsinogen Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In contrast to these small of the heavy chain the of trypsinogen with the catalytic efficiency m of L-BEK was at pH 5.6 and at pH 8.4. heavy chain motifs the low domain and the domain strongly influence the recognition of trypsinogen. The in trypsinogen activation is not by in the of L-BEK is These results the enteropeptidase heavy chain contains an that with to trypsinogen activation, the heavy chain also the of enteropeptidase with macromolecular protease inhibitors. BEK purified from intestine was to be by with a of nm J.J. Light A. J. Biol. Chem. 1979; 254: 1677-1683Abstract Full Text PDF PubMed Google Scholar), and recombinant HL-BEK also was found to be by with a i * of 1.8 nm In the were of was with a in a low was with a K i of 99 and this to the final with a rate of of the heavy chain little on k the final K i * to 6.2 and the K i to nm was for BEK is to J.J. Light A. J. Biol. Chem. 1979; 254: 1677-1683Abstract Full Text PDF PubMed Google Scholar), and HL-BEK also was not by of the heavy chain L-BEK to by with a K i * of 1.6 nm In the of the L-BEK reaction with did not indicate the of an low enteropeptidase light chain by and A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and a recombinant with an A. C. Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) also by Thus, of the heavy chain the of enteropeptidase by but by enteropeptidase and not amino to the that with the catalytic and for this the enteropeptidase heavy chain may be a of reaction with these and other kinetic of the enteropeptidase heavy chain it in the recognition of macromolecular and inhibitors. The activation of trypsinogen is by the heavy and influence is acid that in the The of enteropeptidase to protease also appears to in on the heavy chain. The of the to of for the of serine proteases to macromolecular These serine proteases and to by structural of both catalytic and The activation of trypsinogen is a in the activation of other digestive hydrolases within the lumen of the and of this reaction on all vertebrate activated by proteolytic of a in an amino-terminal peptide that contains the sequence (2Bricteux-Gregoire S. Schyns R. Florkin M. Comp. Biochem. Physiol. B Comp. Biochem. 1972; 42: 23-39Crossref PubMed Scopus (35) Google Scholar). of enteropeptidase that residues on the of the catalytic (light chain) with the residues of trypsinogen activation M. M. S. H. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). may for the recognition of small peptide but not to the recognition of trypsinogen. The light chain has been by of purified bovine enteropeptidase A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or by of recombinant light chain A. C. Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). small synthetic peptides, but trypsinogen. the recognition of small only the light whereas of trypsinogen may also on the heavy chain. in trypsinogen recognition were in enteropeptidase by (6Anderson L.E. Walsh K.A. Neurath H. Biochemistry. 1977; 16: 3354-3360Crossref PubMed Scopus (44) Google Scholar, PubMed Scopus Google Scholar) or by J. S. PubMed Scopus Google Scholar). that the catalytic and at to and to these be on the heavy chain or the light chain. To the of enteropeptidase substrate variants were constructed that or of the heavy chain. of the transmembrane segment appears to little on trypsinogen activation by bovine A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) or M. M. S. H. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) the membrane-spanning domain was with a signal peptide to of secreted recombinant from was to the of the that serine proteases an that the activation peptide to the protease domain, and of enteropeptidase with a of serine proteases that the of the heavy chain is to the light chain or in (5Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (145) Google Scholar). To this and to the of an or the the amino of the heavy chain that to the activation peptide. of the short activation peptide and the catalytic domain was by that pro-L-BEK and L-BEK similar whereas after the apparent mass of L-BEK is than that of pro-L-BEK pro-HL-BEK and pro-L-BEK were purified as single-chain and were found to little catalytic In pro-HL-BEK than the of HL-BEK The activation of proenteropeptidase in the sequence has the of a for by trypsin (5Kitamoto Y. Yuan X. Wu Q. McCourt D.W. Sadler J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7588-7592Crossref PubMed Scopus (145) Google Scholar). In trypsin readily activated both pro-HL-BEK and pro-L-BEK by cleaving after was activated than that the heavy chain has a on the recognition of proenteropeptidase by trypsin. The of proenteropeptidase to were not to of the K m for by be μm not trypsin cleaved pro-HL-BEK at a rate of and pro-L-BEK at a rate of at nm), and these similar to of other trypsin Thus, proenteropeptidase is as a single-chain and it is activated efficiently by in activates the of a be in study is to trypsinogen or proenteropeptidase is for this or protease may be in duodenal mucosa or pancreatic of the heavy chain only a on proenteropeptidase activation, it a and on enteropeptidase substrate specificity. and L-BEK similar kinetic parameters for of that the heavy chain not strongly influence the recognition of small the substrate BEK and HL-BEK also similar m and the cat that the transmembrane and little on trypsinogen is with the that proteolytic of enteropeptidase from did not trypsinogen Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In contrast to these small of the heavy chain the of trypsinogen with the catalytic efficiency m of L-BEK was at pH 5.6 and at pH 8.4. heavy chain motifs the low domain and the domain strongly influence the recognition of trypsinogen. The in trypsinogen activation is not by in the of L-BEK is These results the enteropeptidase heavy chain contains an that with trypsinogen. In to trypsinogen activation, the heavy chain also the of enteropeptidase with macromolecular protease inhibitors. BEK purified from intestine was to be by with a of nm J.J. Light A. J. Biol. Chem. 1979; 254: 1677-1683Abstract Full Text PDF PubMed Google Scholar), and recombinant HL-BEK also was found to be by with a i * of 1.8 nm In the were of was with a in a low was with a K i of 99 and this to the final with a rate of of the heavy chain little on k the final K i * to 6.2 and the K i to nm was for BEK is to J.J. Light A. J. Biol. Chem. 1979; 254: 1677-1683Abstract Full Text PDF PubMed Google Scholar), and HL-BEK also was not by of the heavy chain L-BEK to by with a K i * of 1.6 nm In the of the L-BEK reaction with did not indicate the of an low enteropeptidase light chain by and A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and a recombinant with an A. C. Light A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) also by Thus, of the heavy chain the of enteropeptidase by but by enteropeptidase and not amino to the that with the catalytic and for this the enteropeptidase heavy chain may be a of reaction with these and other inhibitors. The kinetic of the enteropeptidase heavy chain it in the recognition of macromolecular and inhibitors. The activation of trypsinogen is by the heavy and influence is acid that in the The of enteropeptidase to protease also appears to in on the heavy chain. The of the to of for the of serine proteases to macromolecular These serine proteases and to by structural of both catalytic and and of at for amino acid