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.

Fig. 1.

Electrostatic potential map, catalytic scheme, and experimental X-ray structural models of KSI. (A) Electrostatic potential map of the D40N active site generated by solving the Poisson–Boltzmann equation using the program DelPhi (55, 56) and plotted on the solvent-accessible surface of the X-ray structural model for M116C-CN/D40N (apoprotein, PDB ID code 3OXA). Equilenin is modeled in the active site, shown in stick representation, with carbon atoms in green. The foreground residues have been removed to allow viewing into the active site. A drop in potential from 80 mV to −80 mV in the space of two Å equates to an 8 MV/cm field. (B) Reaction mechanism for pKSI catalysis of steroid isomerization, shown for the substrate 5-androstene-3,17-dione. (C) Superposition of the aligned X-ray crystal structures of M116C-CN/D40N, F86C-CN/D40N (equilenin bound, PDB ID code 3OWU) and M105C-CN/D40N (equilenin bound, PDB ID code 3OWY). The carbon atoms of Tyr16, Asp103, Asn40, and the bound ligand equilenin are shown in gray and derived from 3OWU. The carbon atoms of the Cys-CN residue for M116C-CN, M105C-CN, and F86C-CN are shown as green, magenta, and cyan, respectively. Atoms for nitrogen, oxygen, and sulfur appear as blue, red, and gold, respectively, for all structures. Inset: View of the thiocyanate groups of the three constructs translated in the same axis system so that sulfur atoms (yellow) coincide at the same location. The common Cartesian coordinate system is shown with gray sticks, demonstrating the relative orientation of each probe’s bond vectors to the coordinate system of the protein structure. (D) The local environment surrounding each of the three probes is shown in semitransparent surface representation. Note that cavities in the surface result in the nitrile probe being solvent-exposed for M116C-CN and F86C-CN, whereas M105C-CN lies beneath the surface.

Fig. 2.

IR absorption spectra of KSI-CN variants in D40 versus D40N and at pH 6 versus 8. (A–C) X-ray structures of the ionizable groups in the region around each nitrile probe are shown with the Asn at position 40. In gray are the active site ionizable groups (Y32 omitted for clarity). The Cys-CN residue in each variant (carbon atoms colored as in Fig. 1) is aligned according to the axis system shown in the upper left inset of A. Dashed lines indicate the distance from the probe to the side chain of residue 40, which are 11, 9.2, and 5.7 Å, for positions 105, 86, and 116, respectively. (D–F) Absorption spectra of the nitrile stretching region for M105C-CN, F86C-CN, and M116C-CN (from left to right, respectively) in D40 (red) versus D40N (black) at pH 7.1. (G–I) A rotated view of the ionizable groups of D40N. D103 is circled in red in the cases where the observations are most at odds with the expectations for D103 ionization (positions 105 and 86). (J–L) Absorption spectra of the nitrile stretching region for KSI-CN variants at pH 5.6 (red) or pH 7.8 (black) in 100 mM MES buffer.

Fig. 3.

¹³C-NMR Chemical shift versus nitrile stretching frequency for nitrile probes in apo or ligand-bound KSI-CN variants: F86C-CN (blue, -1.6 cm^-1/ppm, R² = 0.9), M116C-CN (black, -2.2 cm^-1/ppm, R² = 0.89), and M105C-CN (red), in solid circles (data from Table 2). These results are compared to data for the model compound ethylthiocyanate in different aprotic solvents, taken from ref. , in green diamonds (-1.7 cm^-1/ppm, R² = 0.68). Hydrogen bond corrections of −13 and -10 cm^-1, applied to the nitrile stretching frequency of F86C-CN and M116C-CN, respectively, as described in the text, give the values shown with blue and black open circles, respectively. For a combined dataset including ethylthiocyanate, M105C-CN, and the corrected F86C-CN and M116C-CN values, the best fit line gives a slope of -1.8 cm^-1/ppm, R² = 0.83. Data points are numbered for ethylthiocyanate in dimethylsulfoxide (1), dimethylformamide (2), acetone (3), methylene chloride (4), tetrahydrofuran (5), chloroform (6), toluene (7), and cyclohexane (8). Data for KSI-CN variants are numbered 9–19: apo M116C-CN/D40N (9), apo M116C-CN (10), M116C-CN/D40N•equilenin (11), M116C-CN/D40N•3-F-4-NO₂-phenol (12), M116C-CN/D40N•4-NO₂-phenol (13), apo F86C-CN (14), apo F86C-CN/D40N (15), F86C-CN/D40N•equilenin (16), apo M105C-CN/D40N (17), apo M105C-CN (18), and M105C-CN/D40N•3-F-4-NO₂-phenol (19).

Fig. 4.

Absorption (A) and Stark (B) spectra for KSI-CN variants. (A) Absorption spectra of the apo enzymes at 77 K in 50% glycerol/water at 10–15 mM, scaled to a common concentration and path-length. From left to right, M105C-CN, M106C-CN, and F86C-CN in red, black, and blue, respectively. (B) Vibrational Stark spectra of the same samples, scaled to a common value of applied field equal to 1 MV/cm. M116C-CN and F86C-CN are further scaled 4X and 8X, respectively, for ease of visualization (broader absorption features necessarily result in smaller features in a Stark spectrum as the Stark spectrum is proportional to the second derivative of the absorption). Analytical frequency-weighted second derivatives of the absorption spectra of A are plotted with dashed lines next to the corresponding Stark spectrum.

Fig. 5.

Comparison of experimental electrostatic field changes (in MV/cm) observed at each probe site and predicted field changes calculated for each probe site based on ionization of different active site residues using DelPhi and the experimental Stark tuning rate. Comparison of experimental versus calculated field changes are made for D40 versus D40N (A–B) and for raising the pH from 6 to 8 (C–D). A and C show the sign and magnitude of the observed (black) and predicted shifts side by side, with predictions for the scenarios in which D103, Y16, Y32, or Y57 (red, green, blue, and magenta, respectively) lose a proton due to mutation or raising the pH. B and D show the magnitude of the error in the predicted shift depending on the choice for the group undergoing ionization at each of the probe sites, 105, 86, and 116 (orange, light blue, and gray, respectively).

Fig. 6.

pH titration of the UV absorbance at 300 nm for pKSI D40N. The molar absorptivity change at 300 nm is plotted relative to the value observed with an external buffer pH of 4.7. A two-stage sigmoidal curve was fit to the data, yielding a pK_a of 6.3 for the lower pK_a. The dashed line shows the molar absorptivity change at 300 nm observed for the tyrosine deprotonation in aqueous buffer. Inset: UV-vis absorption difference spectra for KSI D40N (solid) and aqueous tyrosine (dashed), calculated from the pH 9.1 minus pH 5.5, and pH 14 minus pH 4.7 spectra, respectively.

Fig. 7.

¹³C-NMR spectra for ¹³C_ζ-Tyr-labled KSI. (A–C) apo-WT, apo-D40N and D40N in complex with 4-NO₂-phenolate, respectively. Spectra were taken in 40 mM potassium phosphate, pH 7.1, 23 °C. Chemical shifts for the neutral and ionized tyrosine in small unstructured peptides (58) are shown with dashed lines.

Fig. P1.

Electrostatic potential map, experimental X-ray structural models, and IR spectra of KSI. (A) Electrostatic potential map of the D40N active site generated by solving the Poisson–Boltzmann equation. The transition-state analog equilenin is modeled in the active site in green. Foreground residues have been removed to allow viewing into the active site. (B) Superposition of the aligned X-ray crystal structures of three probe-modified KSI variants in a close-up view of key residues, depicting nitrile groups introduced at positions 105, 116, and 86 in magenta, green, and cyan, respectively. Catalytic residues and their numbering are also shown, along with equilenin, in gray. (C) IR absorption spectra in the CN-stretching region of KSI-CN variants.