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 immunocyto‐chemical 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 α‐N‐tosylglycylprolyllysine 4‐nitroanilide (k_cat/K_m of 35 000 M⁻¹ s⁻¹), α‐N‐succinylthreonylprolyllysme 4‐nitroanilide (k_cat/K_m of 18000 M⁻¹ s⁻¹) and α‐N‐serylprolyllysine 4‐nitroanilide (K_cat/k_m 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 (K_m of 87 μ; k_cat of 1.4 s⁻¹; k_cat/K_m 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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