Determining the role(s) of prime‐side residues in macromolecular inhibition of trypsin‐fold serine proteases (933.2)

Abstract

Trypsin‐fold serine proteases are among the most abundant of proteases. By identifying residues that are important for enzyme‐inhibitor interactions in trypsin, we may advance the development of protease‐based therapies in general. Lysine‐60 (K60) is a highly conserved prime‐side residue in trypsin‐fold serine proteases. In trypsin‐inhibitor co‐crystals, K60 hydrogen bonds to tyrosine‐39 (Y39) and is positioned to restrict conformational mobility of phenylalanine‐41 (F41), possibly optimizing the observed hydrogen bond interactions between inhibitors, Y39 and F41. Substitution of K60 with other 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, K60M, K60R, Y39S, and Y39F and characterized them with respect to their activities and sensitivities to macromolecular inhibitors soybean trypsin inhibitor (SBTI), bovine pancreatic trypsin inhibitor (BPTI) and ecotin, an inhibitor expressed in E. coli. Our initial results show that, compared to wild‐type trypsin, all variants maintain similar catalytic activity, but‐K60A, K60V, K60I, K60M, and Y39F are more sensitive to inhibition while variants K60R and Y39S are less sensitive to inhibition. Collectively, these results suggest that K60 and Y39 do indeed play significant roles in inhibitor binding.Grant Funding Source: This work was supported by NSF