New Strategies for Exploring RNA's 2′-OH Expose the Importance of Solvent during Group II Intron Catalysis

AbstractThe 2′-hydroxyl group contributes inextricably to the functional behavior of many RNA molecules, fulfilling numerous essential chemical roles. To assess how hydroxyl groups impart functional behavior to RNA, we developed a series of experimental strategies using an array of nucleoside analogs. These strategies provide the means to investigate whether a hydroxyl group influences function directly (via hydrogen bonding or metal ion coordination), indirectly (via space-filling capacity, inductive effects, and sugar conformation), or through interactions with solvent. The nucleoside analogs span a broad range of chemical diversity, such that quantitative structure activity relationships (QSAR) now become possible in the exploration of RNA biology. We employed these strategies to investigate the spliced exons reopening (SER) reaction of the group II intron. Our results suggest that the cleavage site 2′-hydroxyl may mediate an interaction with a water molecule

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

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 p<i>K</i>_a 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 p<i>K</i>_a of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional p<i>K</i>_a-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

Correction to “Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid Isomerase”

Correction to “Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid Isomerase

Correction to “Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid Isomerase”

Correction to “Evaluating the Catalytic Contribution from the Oxyanion Hole in Ketosteroid Isomerase

Uncovering the Determinants of a Highly Perturbed Tyrosine p<i>K</i>_a in the Active Site of Ketosteroid Isomerase

Within the idiosyncratic enzyme active-site environment, side chain and ligand p<i>K</i>_a values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of systems has often attributed perturbed p<i>K</i>_a values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a p<i>K</i>_a perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, ¹³C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallography, was used to unravel the basis for this perturbed Tyr p<i>K</i>_a. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its p<i>K</i>_a rather than lowers it; this effect, and part of the increase in the Tyr p<i>K</i>_a from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water molecule positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural analysis can greatly facilitate our understanding of enzyme active-site energetics. The extensive data set provided may also be a valuable resource for those wishing to extensively test computational approaches for determining enzymatic p<i>K</i>_a values and energetic effects