Modifying Trypsin to Weaken Inhibitor Interaction

Serine proteases have been used as therapeutics to help with health maladies such as Christmas Disease, a blood coagulation disorder. They may potentially be administered as drugs in order to aid many other conditions, but their activity is limited by natural regulatory inhibitors which serve to regulate the action of proteolytic enzymes and prevent diseases such as pancreatitis. A number of these inhibitors bind to the hydroxyl group of Y39 in the S1′ pocket of trypsin‐fold serine proteases. We expect that substituting this tyrosine with a leucine will weaken inhibitor interaction, thus increasing the half‐life of serine proteases in the body. Using trypsin as a model serine protease, the variant Y39L was designed via PCR mutagenesis, expressed in P. Pastoris, and purified through hydrophobic interaction chromatography. Before determining if inhibitor interaction with Y39L was weaker than with wild‐type trypsin, we compared their kinetics. In this study, we show that Y39L has comparable activity (kcat = 2.20 × 103 min‐1) and binding capability to Z‐GPR‐pNA (KM = 6.89 ± 1.28 μM) as wild‐type trypsin. Using the commercial competitive inhibitor benzamidine, we also show that the active site of Y39L has not been compromised by this amino acid replacement (KI = 5.28 μM). This implies that if the inhibition of this variant with macromolecular inhibitors is not consistent with that of WT, any difference must be due to the change made in the S1’ site. These studies will be instrumental in the development of trypsin‐fold serine proteases that may potentially be used as therapeutic agents to aid in several biological processes. Support or Funding Information National Institute of Health ‐ MARC National Science Foundation ‐ CSU‐LSAMP