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, Enter opeptidase is synthesized as a single-chain protein, whereas purified enteropeptidase contains a approximate to 47-kDa serine protease domai n (light chain) and a disulfide-linked approximate to 120-kDa heavy ch ain, The heavy chain contains an amino-terminal membrane-spanning segm ent and several repeated structural motifs of unknown function, To stu dy the role of heavy chain motifs in substrate recognition, secreted v ariants of recombinant bovine proenteropeptidase were constructed by r eplacing the transmembrane domain with a signal peptide, Secreted vari ants containing both the heavy chain (minus the transmembrane domain) and the catalytic light chain (pro-HL-BEK (where BEK is bovine enterop eptidase)) or only the catalytic domain (pro-L-BEK) were expressed in baby hamster kidney cells and purified, Single-chain pro-HL-BEK and pr o-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 mu M and k(cat) = 4.0 s(-1)) and by HL-BEK (K-m = 5.6 mu M and k(ca t) = 2.2 s(-1)), but not by L-BEK (K-m = 133 mu M and k(cat) = 0.1 s(- 1)); HL-BEK cleaved trypsinogen at pH 5.6 with 520-fold greater cataly tic efficiency than did L-BEK. Qualitatively similar results were obta ined at pH 8.4. In contrast to this striking difference in trypsinogen recognition, the small synthetic substrate Gly-Asp-Asp-Asp-Asp-Lys-be ta-naphthylamide was cleaved with similar kinetic parameters by both H L-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 influe nced the rate of reaction with protease inhibitors, Bovine pancreatic trypsin inhibitor preferred HL-BEK (initial K-i = 99 nM and final Ki = 1.8 nM) over L-BEK (K-i = 698 nM and K-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 enteropept idase heavy chain has little influence on the recognition of small pep tides, but strongly influences macromolecular substrate recognition an d inhibitor specificity.