Dissecting Proton Delocalization in an Enzyme’s Hydrogen Bond Network with Unnatural Amino Acids

Extended hydrogen bond networks are a common structural motif of enzymes. A recent analysis proposed quantum delocalization of protons as a feature present in the hydrogen bond network spanning a triad of tyrosines (Y¹⁶, Y³², and Y⁵⁷) in the active site of ketosteroid isomerase (KSI), contributing to its unusual acidity and large isotope shift. In this study, we utilized amber suppression to substitute each tyrosine residue with 3-chlorotyrosine to test the delocalization model and the proton affinity balance in the triad. X-ray crystal structures of each variant demonstrated that the structure, notably the O–O distances within the triad, was unaffected by 3-chlorotyrosine substitutions. The changes in the cluster’s acidity and the acidity’s isotope dependence in these variants were assessed via UV–vis spectroscopy and the proton sharing pattern among individual residues with ¹³C nuclear magnetic resonance. Our data show p<i>K</i>_a detuning at each triad residue alters the proton delocalization behavior in the H-bond network. The extra stabilization energy necessary for the unusual acidity mainly comes from the strong interactions between Y⁵⁷ and Y¹⁶. This is further enabled by Y³², which maintains the right geometry and matched proton affinity in the triad. This study provides a rich picture of the energetics of the hydrogen bond network in enzymes for further model refinement