Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide

Enteropeptidase is a membrane-bound serine protease that initiates the acti vation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidy l substrates that resemble trypsinogen activation peptides such as Val-(Asp )(4)Lys. To characterize the determinants of substrate specificity, we solv ed the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 Angstrom resolution in complex with the inhibitor Val-(Asp),Lys-chlorom ethane. The catalytic mechanism and contacts with lysine at substrate posit ion P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the en zyme surface mainly through salt bridges with the N-zeta; atom of Lys99. Mu tation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)(4)-Lys-beta-naphthylamid e and reduced the rate of inhibition by Val-(Asp),Lys-chloromethane 22 to 9 0-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol-l of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen. (C) 1999 Academic Pre ss.