The effect of disrupting a distal hydrogen bond network on serine protease function

Engineered serine proteases constitute a distinct therapeutic class with a potential for diverse clinical applications. One main complication faced when using serine proteases as therapeutic agents is their relatively short half‐life due to the abundance of tight binding inhibitors that target serine proteases to regulate proteolytic activity and rapidly remove them from circulation. However, if a protease is to be effective as a therapeutic agent, it needs to display resistance towards inhibition. Given that the majority of protease therapeutics currently used are of the trypsin fold, trypsin itself serves as an ideal structure‐function relationship model to address this issue. Previous studies, using small synthetic peptides, bovine pancreatic trypsin inhibitor (BPTI) and an ecotin variant (M84R ecotin) with engineered trypsin variants have shown that prime side interactions can affect association with macromolecular inhibitors through interaction with residues at positions 39 and 60. The K60A trypsin variant (K60A‐Tn) had the lowest amount of free enzyme in equilibrium inhibition studies while using BPTI, but the opposite was observed with M84R ecotin. There is evidence that increased mobility of Y39 caused the K60 substitution to impact P4′ interactions and P2′ hydrogen bond, resulting in altered trypsin:inhibitor interactions. Specifically, replacing lysine‐60 with alanine does not allow the K60 and Y39 charged hydrogen bond interaction regardless of the P4′ hydrogen bond presence, suggesting that a polymorphism at position 60 may influence interaction between Y39 and inhibitors. However, given that BPTI and ecotin are also proteins, their interaction with trypsin likely reflects the interaction that serine proteases will have with natural protein substrates. Therefore, a desired weakened interaction between the engineered trypsin and the inhibitor may also suggest a similar weakened interaction between the engineered trypsin and protein substrates which cannot be observed with the small peptide substrates that were used previously. Our focus in this study is to use a protein as a substrate, anticipating insight on the enzyme‐substrate interaction via catalytic activity of K60A‐Tn compared to wild‐type trypsin. Without the K60 and Y39 charged hydrogen bond interaction, we predict to see weakened substrate affinities, causing lowered catalytic efficiency. Support or Funding Information Dr. Teaster Baird National Institute of Health (NIH)