Tyrosine sulfation of human trypsin steers S2' subsite selectivity towards basic amino acids.

ABSTRACT

Human cationic and anionic trypsins are sulfated on Tyr154, a residue which helps to shape the prime side substrate-binding subsites. Here, we used phage display technology to assess the significance of tyrosine sulfation for the specificity of human trypsins. The prime side residues P1'-P4' in the binding loop of bovine pancreatic trypsin inhibitor (BPTI) were fully randomized and tight binding inhibitor phages were selected against non-sulfated and sulfated human cationic trypsin. The selection pattern for the two targets differed mostly at the P2' position, where variants selected against non-sulfated trypsin contained primarily aliphatic residues (Leu, Ile, Met), while variants selected against sulfated trypsin were enriched also for Arg. BPTI variants carrying Arg, Lys, Ile, Leu or Ala at the P2' position of the binding loop were purified and equilibrium dissociation constants were determined against non-sulfated and sulfated cationic and anionic human trypsins. BPTI variants harboring apolar residues at P2' exhibited 3-12-fold lower affinity to sulfated trypsin relative to the non-sulfated enzyme, whereas BPTI variants containing basic residues at P2' had comparable affinity to both trypsin forms. Taken together, the observations demonstrate that the tyrosyl sulfate in human trypsins interacts with the P2' position of the substrate-like inhibitor and this modification increases P2' selectivity towards basic side chains.