How the Loss of a Single Hydroxyl Group Affects the Proteolytic Activity of the Model Serine Protease, Trypsin

Abstract

Trypsin is used as a model for the study of serine proteases, for its fold is commonly observed in this family of enzymes. The overall aim of this study is to discern whether the Y39F trypsin variant will act on a macromolecular substrate similar to the way it interacts with macromolecular inhibitors, specifically focusing on the S prime (S') side. In wild‐type trypsin, the hydroxyl group in tyrosine at position 39 serves to stabilize the interaction with macromolecular inhibitors via the formation of hydrogen bonds at the P prime (P') subsites. A Y39F substitution was made to reduce the affinity between the enzyme variant and inhibitor. A phenylalanine was used in place of the tyrosine because the two amino acid residues are chemically similar with the exception of the hydroxyl functional group missing from the phenylalanine. The absence of the hydroxyl functional group serves to prevent the formation of hydrogen bonds between macromolecular substrates that interact with the S' site of the enzyme, presumably weakening of the enzyme‐inhibitor interaction. However, previous research from our group demonstrated that the Y39F trypsin variant was more sensitive to inhibition from both bovine pancreatic trypsin inhibitor (BPTI) and ecotin compared to wild‐type trypsin. Molecular modeling suggests that the tighter binding is a consequence of the formation of a hydrogen bond between the P2′ site and the histidine side chain at position 40 not seen in wild type trypsin. The presence of the newly formed hydrogen bond in the Y39F trypsin variant helps stabilize the enzyme‐inhibitor complex. Still, Y39F trypsin retained similar catalytic activity against N‐α‐benzyloxycarbonyl‐glycylprolylarginine p‐nitroanilide (Z‐GPR‐pNA). However, Z‐GPR‐pNA physically cannot interact with the modified S' subsites as the macromolecular inhibitors, and likely macromolecular substrates, do. Therefore, the activity observed with Z‐GPR‐pNA may not accurately reflect the activity of the Y39F variant towards macromolecular substrates. In this study, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and gel imaging to monitor and compare the progression of macromolecular substrate hydrolysis of Y39F and wild‐type trypsin. Based on the observations made with BPTI and ecotin, we would expect that the Y39F variant would bind more tightly to a macromolecular substrate as well, likely increasing the efficiency of cleavage relative to wild‐type.Support or Funding InformationNSF Award MCB‐0643988‐02