Catalog Number: 199800

CAS #: 9014-74-8


Molecular Weight/Structure: 147,000-150,000 heterodimer consisting of 115-135 kD and 35-47 kD subunits. Two disulfide bridges ling the light and heavy chains. The light chain is a trypsin-like serine enzyme, which contains the whole set of amino acids necessary for the formation of both the catalytic center and the tetraaspartyl binding site. However, this is not sufficient for highly effective hydrolysis of the natural substrate, trypsinogen.1

E.C. #

Synonym: Enteropeptidase

Form: Solution

Unit Definition:

(One FLAGBAP unit is equal to 10X the activity of a standard trypsinogen unit (Liepnieks and Light, 1979).)

Description: Enterokinase is a highly specific serine protease. It is a glycoprotein containing 35% carbohydrate. By activating trypsinogen, Enterokinase catalyzes hydrolysis of the polypeptide chain only after an -(Asp)4-Lys- sequence.

It was found that calcium ions regulate the Enterokinase activity.1 A type of intramolecular autolysis resulting from the loss of these ions causes a considerable decrease in the Enterokinase catalytic activity toward trypsinogen, whereas the activity toward synthetic low- and high-molecular-mass substrates containing the tetraaspartyl-lysine linker is retained. The localization of the autolysis degradation sites suggested the presence of a secondary substrate-binding center for trypsinogen in the N-terminal fragment 118-465 of the enzyme heavy chain.1

The high specificity and intricate structure of Enterokinase determine its relations with protein inhibitors. It has been shown that the bovine deudenum contains a natural kunin-like inhibitor of enterokinase (DI-9), which may be involved in the regulation of calcium-dependent autolysis of the heavy enzyme chain. As a result, a truncated form, which is low active toward trypsinogen, forms. It has also been suggested that in the presence of, in certain regions of the heavy chain, one or several calcium binding sites and the regulation of the Enterokinase activity by calcium ions as well as by the inhibitor DI-9.1

Synthetic Substrate: Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, MP catalog number 157235

Recognition Peptide:


* Peptides are resistant to cleavage if proline occupies the X position.

Inhibition: Inhibited by soybean trypsin inhibitor.

Typical Use:

Activity Assay:



Step 1
Pipette the following reagents into suitable tubes:
Test Mix
Blank Mix
Reagent A (Buffer)
1.80 ml
1.80 ml
Reagent C (Trypsinogen)
0.50 ml
0.50 ml
Mix by inversion and equilibrate to 25C. Then add:
Reagent D (Enterokinase)
0.10 ml
0.00 ml
Deionized water
0.00 ml
0.10 ml
Immediately mix by inversion and incubate at 25C for exactly 15 minutes. Then add:
Reagent G (HCl-CaCl2)
3.00 ml
3.00 ml
Step 2
Pipette the following reagents into suitable quartz cuvettes:
Reagent F (BAEE)
3.00 ml
3.00 ml
Equilibrate to 25C. Monitor the A253nm until constant, using a suitably thermostatted spectrophotometer. Then add:
Text Mix from step 1
0.20 ml
0.00 ml
Blank Mix from step 1
0.00 ml
0.20 ml

Immediately mix by inversion and record the increase in A253nm for approximately 5 minutes. Obtain the DA253nm/minute using the maximum linear rate for both the Test and Blank.



Final Assay Concentration:

In a 2.40 ml reaction mix, the final concentrations are 30 mM succinate, 1 mM calcium chloride, 0.2 mM hydrochloric acid, 0.5 mg trypsinogen and 0.2 - 0.5 unit Enterokinase.


Catalog NumberDescriptionSize
199800Enterokinase from calf intestinal mucosa1 KU
5 KU

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Laboratory of Chemistry of the Proteolytic Enzymes, Web Site (2003 with last modification of Feb 14, 2002).
  2. Baird, T., et al., "Generation of active trypsin by chemical cleavage." Tetrahedron, v. 56:48, 9477-9485 (2000).
  3. Baratti, J., Maroux, S., Louvard, D. and Desnuelle, P., Biochimica et Biophysica Acta, v. 315, 147-161 (1973).
  4. Fukuoka, S.-I. and Nyaruhucha, C.M., "Expression and functional analysis of rat P23, a gut hormone-inducible isofrom of trypsin, reveals its resistance to proteinaceous trypsin inhibitors." Biochimica et Biophysica acta/Molecular Basis of Disease, v. 1588:2, 106-112 (2002).
  5. Grant, D.A.W. and Hermon-Taylor, J., Biochem. J., v. 147, 363-366 (1975).
  6. Holzinger, A., et al., "Mutations in the proEnterokinase gene are the molecular cause of congenital Enterokinase deficiency." Am. J. Hum. Genet., v. 70, 20-25 (2002).
  7. Ichishima, E., "Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries." Biosci. Biotechnol. Biochem., v. 64:4, 675-688 (2000).
  8. Liepnieks, J. and Light, A., J. Biol. Chem., v. 254, 1677-1683 (1979).
  9. Lu, D.S., et al., "Crystal structure of Enterokinase light chain complexed with an analog of the trypsinogen activation peptide." J. Mol. Biol., v. 292, 361-373 (1999).
  10. Matsushima, M., et al., "Purification and further characterization of Enterokinase from porcine duodenum." J. Biochem. (Tokyo), v. 125:5, 947-951 (1999).
  11. Mikhailova, A.G. and Rumsh, L.D., "Autolysis of bovine Enterokinase heavy chain: evidence of fragment 118-465 involvement in trypsinogen activation." FEBS Letters, v. 442:2-3, 226-230 (1999).
  12. Mikhailova, A.G. and Rumsh, L.D., "Enterokinase - Structure, function, and application in biotechnology." Appl. Biochem. Biotechnol., v. 88, 159-174 (2000).
  13. Moroz, S.P., et al., "Celiac disease in a patient with a congenital deficiency of intestinal Enterokinase." Am. J. Gastroenterol., v. 96, 2251-2254 (2001).
  14. Shibanova, E.D., et al., "Specific features of Enterokinase hydrolysis of chimeric proteins at the specific linker (Asp)4Lys depending on the refolding conditions." Russ. J. Bioorg. Chem. (Engl. Transl.), v. 26:7, 466-473 (2000).
  15. Song, H.W., Choi, S.I. and Seong, B.L., "Engineered recombinant Enterokinase catalytic subunit: Effect of N-terminal modification." Arch. Biochem. Biophys., v. 400, 1-6 (2002).
  16. Zheng, X.L., Lu, D.S. and Sadler, J.E., "Apical sorting of bovine Enterokinase does not involve detergent-resistant association with sphingolipid-cholesterol rafts." J. Biol. Chem., v. 274, 1596-1605 (1999).
  17. Zheng, X.L. and Sadler, J.E., "Mucin-like domain of Enterokinase directs apical targeting in Madin-Darby canine kidney cells." J. Biol. Chem., v. 277, 6858-6863 (2002).