Determining the role(s) of prime‐side residues in macromolecular inhibition of trypsin‐fold serine proteases

Abstract

Trypsin‐fold serine proteases are among the most abundant of all proteases. By identifying residues that are important for enzyme‐inhibitor interaction in trypsin, we may advance the development of protease‐based therapies in general. Lysine‐60 (K60) is a highly conserved prime‐side residue that may play many roles in trypsin. In co‐crystals, K60 hydrogen bonds to tyrosine‐39 (Y39) and is positioned to restrict conformational mobility of phenylalanine‐41 (F41), possibly limiting the observed hydrogen bond interactions between those residues and macromolecular inhibitors. Substitution of K60 with other amino acid residues may disrupt these interactions and provide insight to their significance with respect to inhibitor binding. To test this hypothesis, we created trypsin variants K60G, K60A, K60V, K60I, and K60R and characterized them with respect to their activities and sensitivities to the macromolecular inhibitors soybean trypsin inhibitor (SBTI) and bovine pancreatic trypsin inhibitor (BPTI). Our initial results show that, compared to wild‐type trypsin, K60A and K60V are catalytically indistinguishable, more resistant to autolysis and more sensitive to inhibition by SBTI and BPTI. Further experiments with ecotin and other trypsin variants can provide additional insight on specific interactions. This work was supported by NSF CAREER Award MCB‐0643988–02 and NIH MARC T34‐GM008574.