Abstract
A preparative method for purification of enteropeptidase (enterokinase) (EC 3. 4. 21.9) is developed. A highly purified form of this enzyme is stabilized by calcium ions and does not contain any other proteolytic enzyme contaminations. These enteropeptidase preparations were successfully used for cleavage of a variety of fusion proteins containing the tetraaspartyl-lysyl sequence in an arbitrary position on the polypeptide chain. A series of substrates was methodically studied, which resulted in the suggestion that the peptide and fusion protein substrates (K m -200μM and 125μM, respectively) were bound to the enzyme through the linker (Asp)4 Lys at the binding site on the light chain of enteropeptidase. Much more efficient hydrolysis of the natural substrate trypsinogen (K m=2.4μM) testifies to a significant contribution of other sites of the substrate and the enzyme in productive binding Avariation in the enzyme's uniquespecificity wasshown to be a result of the autolysis caused by the loss of calcium ions; the cleavage sites were determined. The truncated enzyme containing the C-terminal fragment 466–800 of its heavy chain and the intact light chain does not distinguish the natural substrate trypsinogen, fusion protein, or peptide substrates. These 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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Abbreviations
- BPI:
-
basic bovine protease inhibitor
- STI:
-
soybean trypsin inhibitor
- MPG-PA-2000:
-
porous glass chemically coated with poly (4-nitrophenyl acrylate)
- OG:
-
n-octyl-β-d-glucopyranoside
- BAPNA:
-
α-N-bensotyl-dl-arginine 4-nitroanilide
- GD4K-NA:
-
Gly-Asp-Asp-Asp-Asp-Lys-β-naphthylamide
- Gdn-Bz-ONp:
-
4′-guanidinobenzoate 4-nitrophenyl ester
- Gdn-Bz-OMum:
-
4′-guanidinobenzoate 4-methylumbelliferil ester
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Mikhailova, A.G., Rumsh, L.D. Enteropeptidase. Appl Biochem Biotechnol 88, 159–174 (2000). https://doi.org/10.1385/ABAB:88:1-3:159
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DOI: https://doi.org/10.1385/ABAB:88:1-3:159