Using Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site

Abstract

Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of traditional site-directed mutagenesis experiments. It has been proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization via “short, strong” or “low-barrier” hydrogen bonds that are formed due to matching of their pKa or proton affinity to that of the transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic environments that result in larger energetic responses (i.e., greater “sensitivity”) to ground-state to transition-state charge rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test these models, we substituted tyrosine 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. We found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pKa of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional pKa-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic environment that greatly enhances the energetic response to charge accumulation, consistent with prior experimental results.

A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase

“…We note that the p K a perturbations introduced by Cl-Y incorporation in these mutants are sufficient to impact the formation energy of a presumably strong H-bond interaction between the ligand and the enzyme, as suggested by a systematic change in their rate constants. 23,24 We first rationalize our strategy to quantify the catalytic benefit from the differential proton affinity match. The proton affinity of each KSI variant’s active site (p K α E ) is defined in terms of the solution p K a of a ligand that upon binding forms a 50:50 mixture of protonated and deprotonates states (this is denoted differently from the active site’s p K a because the active site cannot exchange protons with solvent when a ligand is bound).…”

Electric Fields and Enzyme Catalysis

“…Amber suppression and protein semisynthesis enables the installation of more minute mutations than afforded by the 20 canonical proteinogenic amino acids. Studies by Wu & Boxer (119) and Natarajan et al (120) explored the effect of replacing the critical Tyr16 residue with chlorosubstituted and fluorosubstituted tyrosines, respectively. These mutants perturbed KSI’s rate by up to 1.1-fold (for F) and 3.8-fold (for Cl), showing that KSI has no special need to match its substrate’s p K a to act as a proficient catalyst.…”

It Is Important to Compute Intramolecular Hydrogen Bonding in Drug Design?

“…The equilibrium constant K HB for the hydrogen bond acceptor can be measured and converted into pK HB (equations 12-13) and also for the hydrogen bond donor (equations [14][15]. Increasing the polarity of either or both the donor and acceptor will increase the pK HB value.…”

“…Hydrogen (H)-bonds are of immense importance in biological systems [9,10,11] and ubiquitous in nature, playing an important role in protein folding [12], protein-ligand interactions [13], and catalysis [14,15] Despite extensive investigations, there remain many challenges that prevent us from completely understanding how H-bonds modulate molecular function. For that reason, hydrogen bonding has been one of the most interesting areas of research, primarily due to its crucial role in governing the shapes, properties, and functions of biomolecules.…”