Purification and specificity of porcine enterokinase.

Enterokinase (enteropeptidase, EC 3.4.4.8) has been purified from porcine duodenal extracts by chromatography on DEAE-cellulose, carboxymethyl cellulose, Sephadex G-100, and Sephadex G-200. The final product was found to be homogeneous by a number of tests. Its specific activity against trypsinogen (nanomoles of trypsinogen activated per 30 min per mg) was 6550, which represented a 650-fold purilication over the first clear extract. The preparation was still contaminated, however, by traces of aminopeptidase activity. Porcine enterokinase activates bovine trypsinogen much better than does bovine trypsin (K, 6 times lower and koat 2000 times higher). This property arises from the fact that the specificity site of enterokinase recognizes in trypsinogen not merely the basic residue of the -Lys-Ilebond (residues 6 and 7) which is split during activation of the zymogen but also recognizes the sequence -Asp,-Lys (residues 2 to 6) which is present in all of the trypsinogens so far studied. By contrast, aspartyl residues are known to slow down the action of trypsin on the zymogen (autoactivation). The sequence -Asp,,-Lys appears to be essential for the interaction of enterokinase with peptide or protein substrates and inhibitors. For example, the enzyme does not cleave the peptide Val-Alaz-Lys-Ile-Val-Gly and it does not activate chymotrypsinogen A. It does not hydrolyze the fully denatured S-carboxymethyl derivative of chymotrypsinogen and it cleaves exclusively the -Lys-Ilebond (residues 6 and 7) in S-carboxymethylated bovine trypsinogen. Moreover, enterokinase is not inhibited by relatively high concentrations of soybean trypsin inhibitor or the two pancreatic inhibitors described by Kunitz and Kazal. On the other hand, enterokinase is similar to trypsin in several respects. For example, it is inhibited by oc-tosyllysine chloromethyl ketone and by diisopropylphosphorofluoridate, which suggests that an active serine and a histidine residue participate in the catalytic action of enterokinase. Moreover, enterokinase cleaves the synthetic trypsin substrates benzoylarginine ethyl ester and tosylarginine methyl ester. This probably means that the specificity site of enterokinase is composed of several subsites. One, possibly similar to that of trypsin, is responsible for the binding of low molecular weight basic substrates and inhibitors. The