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 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.
Department of Biochemistry, Stanford University School of Medicine , Stanford, California 94305, United States.
Aditya
A
Natarajan
Jason P
JP
Schwans
Daniel
D
Herschlag
D000596:Amino Acids / Q000737:chemistry*; D000838:Anions; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D007664:Ketosteroids / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013251:Steroid Isomerases / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D000838:Anions; D007664:Ketosteroids; D013251:Steroid Isomerases