The Effect of A Y39A Substitution in Trypsin And Its Interaction with Macromolecules ‐ Implications for The Development of Protease Therapeutics

The majority of serine proteases that are used as therapeutic agents, such as tissue and urinary plasminogen activators, share the structural scaffold of trypsin. Trypsin is one of the most well‐studied proteases and is quintessential as a model for examining serine protease interactions with substrates. Previous work has shown that positions 39 and 60 play important roles in inhibitor‐binding to trypsin; an amino acid change results in a decreased affinity due to disrupted prime‐side, backbone interactions. A single amino acid substitution of alanine for tyrosine at position 39 in trypsin (Y39A‐Tn) was found to be less sensitive to inhibitors and has the potential to improve the protease half‐life. In Y39A‐Tn, hydrogen bonding with lysine 60 (K60) on trypsin and with the inhibitor residue at the P4′ position is prevented. Position Y39 of trypsin is well‐conserved among trypsin‐fold serine proteases and its resistance to naturally occurring inhibitors could suggest the same effect for interactions with a protein substrate. To examine the overall catalytic activity toward such a substrate, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and gel imaging were used to analyze digestion product band intensities. Preliminary data suggests that the Y39 variant has similar proteolytic activity towards protein substrates as wild‐type trypsin. Due to increased inhibitor resistance, Y39A variant can serve as a good model for other proteases in the therapeutics field.