Quantitative, directional measurement of electric field heterogeneity in the active site of ketosteroid isomerase

Abstract

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...

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

“…As previously reported by Fafarman et al (20), the 13 C spectrum of unliganded pKSI D40N displays four well-resolved C ζ -Tyr peaks (Fig. 3A, lowest spectrum).…”

“…The 1.2-ppm chemical shift dispersion for the discrete C ζ -Tyr peaks is within the range of 1-2 ppm expected for differential shielding contributions arising from the unique local structural environment of each Tyr (27). Binding of phenols with pK a values increasing from 7.1 to 10.4 resulted in no changes in the position of the most up-field peak at 157.3, previously assigned via mutagenesis to the surface Y119 (20), but steadily shifted the position of the remaining active site Tyr peaks further down-field ( Fig. 3 A and B).…”

“…S2), strongly suggesting that D103 is not the residue that ionizes as bound phenols become protonated. Based on this result and on our prior observation of an ionized Tyr in unliganded pKSI D40N (20), we considered Y16 the most likely residue to be ionized when the bound phenol is neutral. The results described below provide evidence for ionization of both Y16 and Y57.…”

“…A homologous series of bound phenols or naphthols bearing different electron-withdrawing substituents provides a deft experimental tool with which to incrementally vary the proton affinity and negative charge density of the phenolic oxygen (16). These changes tune the structure and strength of hydrogen bonds formed to phenolic ligands (17)(18)(19), and thus provide a systematic probe of the physical and energetic properties of the hydrogen bond network within the KSI oxyanion hole (6-9, 11, 13) and the response of the surrounding protein matrix to such changes (20,21).…”

“…3A, lowest spectrum). The far down-field peak at 165.1 ppm, indicative of an ionized Tyr, was tentatively assigned to Y57 based on comparisons of data and computational models of electrostatic field effects (20). On binding of 4-NO 2 -phenol (pK a = 7.1), which is fully ionized in complex with pKSI D40N (8), all the observed 13 C-Tyr peaks in D40N are shifted up-field to less than 159 ppm (20), as expected for the protonated forms of all four Tyr residues (Fig.…”