A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

Understanding the electrostatic environment within the idiosyncratic interior of folded proteins and its connection to biomolecular function remains a key challenge in biochemistry and biophysics. Vibrational probes incorporated into proteins on specific residues or ligands are exquisitely sensitive reporters of the local environment and how it is altered by pH changes, mutations, structural perturbations, or variations in bound ligands1 -6. While IR frequency shifts associated with various environments can be considered at a qualitative level (e.g., is the probe buried or on a solvent exposed surface?7 , 8), our goal has been to extract quantitative information on electric fields in proteins1 , 2. Although several studies have suggested a connection between observed vibrational band shifts and local electrostatic fields due to the organized environment around the probe3 , 6, in the absence of an independent experimental test it remains uncertain whether these shifts are due principally to electrostatic effects or are dominated by contributions from specific chemical interactions9, such as hydrogen bonds. We report herein a method for identifying and quantifying departures from an electrostatic mechanism for nitrile vibrational shifts that utilizes the relationship between IR frequency shifts and 13 C NMR chemical shifts and demonstrate its utility in a protein.The vibrational Stark effect (VSE) provides the connection between observed IR frequency shifts, (in cm -1 ), and the difference in the local electrostatic field, (in MV/ cm), experienced by a probe at two different sites in a protein or as a result of a pH change, mutation, or ligand binding1 , 2. The sensitivity of an oscillator to electrostatic field is the Stark tuning rate, [in cm -1 /(MV/cm)], which is obtained by measuring the effect of an applied electric field on the IR spectrum11 -13. The nitrile stretch offers an optimal combination of oscillator strength, frequency, and Stark tuning rate for measurements in biological systems11 -14. In this case, observed frequency shifts, , can be used to obtain information on variations in the projection of the protein electrostatic field on the probe through:(1)

Understanding the electrostatic forces and features within highly heterogeneous, anisotropic, and chemically complex enzyme active sites and their connection to biological catalysis remains a longstanding challenge, in part due to the paucity of incisive experimental probes of electrostatic properties within proteins. To quantitatively assess the landscape of electrostatic fields at discrete locations and orientations within an enzyme active site, we have incorporated site-specific thiocyanate vibrational probes into multiple positions within bacterial ketosteroid isomerase. A battery of X-ray crystallographic, vibrational Stark spectroscopy, and NMR studies revealed electrostatic field heterogeneity of 8 MV∕cm between active site probe locations and widely differing sensitivities of discrete probes to common electrostatic perturbations from mutation, ligand binding, and pH changes. Electrostatic calculations based on active site ionization states assigned by literature precedent and computational pK a prediction were unable to quantitatively account for the observed vibrational band shifts. However, electrostatic models of the D40N mutant gave qualitative agreement with the observed vibrational effects when an unusual ionization of an active site tyrosine with a pK a near 7 was included. UV-absorbance and 13 C NMR experiments confirmed the presence of a tyrosinate in the active site, in agreement with electrostatic models. This work provides the most direct measure of the heterogeneous and anisotropic nature of the electrostatic environment within an enzyme active site, and these measurements provide incisive benchmarks for further developing accurate computational models and a foundation for future tests of electrostatics in enzymatic catalysis. E xtensive structural studies of enzymes have revealed that biological catalysis occurs within sequestered active site crevices that solvate reacting substrates with an anisotropic and chemically complex constellation of charged, polar, and hydrophobic groups. But beyond visualization of the chemical composition and architecture of active sites permitted by the rich library of available protein structures, our understanding of the electrostatic nature and properties of this highly heterogeneous environment and its role in molecular recognition and catalysis is largely based on simulations. Computations using three-dimensional structures routinely provide electrostatic potentials, as in Fig. 1A. These potentials have been used to identify and characterize protein-protein and protein-ligand binding sites, and interaction energies derived from these calculations have suggested key contributions from the electrostatic environment to enzymatic rate enhancement and specificity (1-5). However, even the most sophisticated computational methods have multiple approximations and limitations (6-8) and, most importantly, have not been rigorously evaluated through cycles of nontrivial computational predictions and experimental tests. Incisive and quantitative experimental measures o...

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Hydrogen bonds play major roles in biological structure and function. Nonetheless, hydrogen-bonded protons are not typically observed by X-ray crystallography, and most structural studies provide limited insight into the conformational plasticity of individual hydrogen bonds or the dynamical coupling present within hydrogen bond networks. We report the NMR detection of the hydrogen-bonded protons donated by Tyr-42 and Glu-46 to the chromophore oxygen in the active site of the bacterial photoreceptor, photoactive yellow protein (PYP). We have used the NMR resonances for these hydrogen bonds to probe their conformational properties and ability to rearrange in response to nearby electronic perturbation. The detection of geometric isotope effects transmitted between the Tyr-42 and Glu-46 hydrogen bonds provides strong evidence for robust coupling of their equilibrium conformations. Incorporation of a modified chromophore containing an electron-withdrawing cyano group to delocalize negative charge from the chromophore oxygen, analogous to the electronic rearrangement detected upon photon absorption, results in a lengthening of the Tyr-42 and Glu-46 hydrogen bonds and an attenuated hydrogen bond coupling. The results herein elucidate fundamental properties of hydrogen bonds within the complex environment of a protein interior. Furthermore, the robust conformational coupling and plasticity of hydrogen bonds observed in the PYP active site may facilitate the larger-scale dynamical coupling and signal transduction inherent to the biological function that PYP has evolved to carry out and may provide a model for other coupled dynamic systems.charge delocalization ͉ hydrogen bond coupling ͉ protein structure ͉ signal transduction