Dissecting the physical and energetic properties of active site hydrogen bonds in ketosteroid isomerase [electronic resource]
Keywords: Biochemistry.

Hydrogen bonds are a ubiquitous feature of enzyme active sites, stabilizing charge rearrangements on substrate groups over the course of a reaction. Although their importance is clear from traditional site-directed mutagenesis, understanding the origin of their catalytic contribution relative to hydrogen bonds made in aqueous solution remains challenging, in part because traditional mutagenesis ablates hydrogen bonding groups or replaces them with hydrophobic side chains, rendering comparisons between wild type and mutant enzymes complex. Additional complexity arises from the extraordinary sensitivity of hydrogen bond energetics to the surrounding environment. In this thesis, I describe how I substituted tyrosine (Tyr) with fluorotyrosines (F-Tyr's) in the ketosteroid isomerase (KSI) oxyanion hole to systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects and assessed the physical and energetic consequences of this perturbation to provide powerful experimental tests of the behavior of intact hydrogen bonds within an enzyme active site. I observed that a 40-fold increase in F-Tyr acidity caused no significant change in activity for reactions with three different substrates. UV/Vis absorbance and proton NMR spectra of F-Tyr-substituted KSI variants with bound transition state analogs showed that the proton affinity of the tyrosyl group and the physical properties of the Tyr hydrogen bond vary with fluoro-substitution, as expected. Additionally, the change in NMR chemical shift observed was the same as that previously observed in other solvents, providing strong evidence for the effects of fluoro-substitution not being muted by the active site environment. I found that the physical effects of F-Tyr substitution were propagated to Asp103, the other oxyanion hole residue, such that as the Tyr hydrogen bond shortens, the Asp103 hydrogen bond lengthens. This physical coupling could have masked an intrinsic steep energetic sensitivity of the Tyr hydrogen bond to charge accumulation, relative to aqueous solution, resulting in the shallow dependence of catalytic rate on F-Tyr acidity that was observed. To test this alternative model, I determined the dependence of enzymatic activity on F-Tyr acidity with Asp103 mutated to Asn or Gly, and observed the same shallow dependence as before. The observed shallow slopes provide strong evidence that the sensitivity of hydrogen bond energetics to charge accumulation within the KSI active site is not substantially greater than the low sensitivity in aqueous solution and suggest that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional "short, strong" hydrogen bond. These results are consistent with the modest but important overall catalytic contribution of the oxyanion hole residues of ~1000-fold relative to a 'pond mutant' in which these and surrounding residues are removed and are consistent with an important role of positioning of these residues by the enzyme scaffold and surrounding side chains. I close with laying out the key challenges that lie ahead in quantitatively understanding the origin of the enormous rate enhancements that enzymes provide.