Dissecting Proton Delocalization And The Electrostatic Contribution To Catalysis In An Enzyme'S Hydrogen Bond Network With Unnatural Amino Acids

Extended hydrogen bond networks are a common structural motif of enzymes. To understand the functional relevance of the H-bond network in catalysis, this study focuses on a model protein, ketosteroid isomerase (KSI), whose H-bond network in the active site has been shown to promote both proton delocalization across the enzyme-intermediate complex and a strong electric field between the enzyme and the substrate/inhibitor bond involved in catalysis. By utilizing amber suppression, we constructed a series of KSI mutants where the active site H-bond network is preserved while the proton affinity and electrostatic character of individual key residues are selectively perturbed. This allowed us to approach the question from three aspects: how each active site residue contributes to the proton delocalization equilibrium; how changes in the proton delocalization are reflected in the electrostatic interaction between the enzyme and the ligand; and how the degree of proton delocalization and the strength of the electric field affect the catalytic properties of the enzyme. X-ray crystal structures of the variants alone and bound with an intermediate analog demonstrate that the active site geometry is preserved. We quantified the changes in proton delocalization equilibrium with UV-vis spectroscopy, 13C-NMR, and 1H-NMR. In parallel, we also measured the electrostatic interaction between the enzyme and a carbonyl group on an inhibitor where charge separation occurs during catalysis with vibrational Stark effect spectroscopy. The correlations between the extent of proton delocalization, the strength of electric field, and the catalytic efficiency will be discussed to address the contributions of proton delocalization and electrostatic stabilization in this enzymeu0027s catalytic mechanism.