Figure 2.

Semisynthesis of KSI with substituted tyrosines in the oxyanion hole. (A) Structures, abbreviations, and side chain pK_a values of inserted tyrosines. The pK_a values are for the corresponding phenols.^86,87 (B) Substituted tyrosines were protected and incorporated into a 23-mer synthetic peptide with a C-terminal thioester. The remainder of the protein was expressed as a SUMO fusion, followed by removal of the N-terminal SUMO tag and ligation to the synthetic peptide to generate full-length protein. (C) Electrospray mass spectra of semisynthetic (black) and recombinant (gray) WT enzyme (i), and semisynthetic enzymes with substituted tyrosines (ii–v). The expected masses for KSI with Tyr, 2-F- or 3-F-Tyr, 2,6-F₂-Tyr, and 3-Cl-Tyr are shown as brown, red, blue, and green dashed lines.