Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase

Abstract

Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ(5)-3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.

Fig. 1.

Structural model of the active site of KSI bound to C183 and equilenin. (A) Superimposed structures of KSI variants containing a nitrile at positions 116 (C116-¹³C¹⁵N), 17 (C17-¹³C¹⁵N), 86 (C86-¹³C¹⁵N), or 105 (C105-¹³C¹⁵N) bound to equilenin (transparent yellow). C183 (blue) is aligned with equilenin in the superimposed structure. The structure was drawn from Protein Data Bank files 3OXA, 3OX9, and 3OWY using the program PyMOL. The structural model of KSI with a nitrile at position 17 (I17C-¹³C¹⁵N) was created using PyMOL. (B) The movement of electronic charge during the enzymatic reaction of KSI is compared to the movement of electronic charge during the photoexcitation of C183 (C). The red arrow in B and C show the direction of movement of electronic charge when moving from KSI–intermediate complex to KSI–substrate complex. An electronic difference dipole () of approximately 20.2 debye is created from the movement of a single electronic charge over a distance of 4.2 Å during the passage from KSI–intermediate complex to KSI–substrate complex, which is similar in magnitude to  debye created during photoexcitation of enzyme-bound C183 (see text).

Fig. 2.

Steady-state FTIR spectra of ¹³C¹⁵N-labeled mutant forms of KSI. In each panel, the green and blue spectra denote the absorption frequency of -¹³C¹⁵N attached to the sole thiol of the indicated mutant variant of KSI in the apo form and in C183 bound form, respectively.

Fig. 3.

Time-resolved transient IR absorption spectra of ¹³C¹⁵N-labeled mutant forms of KSI after the photoexcitation of bound C183 at 400 nm. A–C, Inset, shows the mean IR-absorption frequency of -¹³C¹⁵N attached to the sole thiol of the indicated mutant form of KSI, at different times after the photoexcitation of the enzyme-bound C183, in ground-state bleach (blue triangles) and electronic excited state (dark-red circles). The solid blue and red line through the data in the inset is a fit to a straight line equation.

Fig. 4.

Polarization-dependent transient IR absorption spectra of ¹³C¹⁵N-labeled mutant forms of KSI at time zero. Transient spectra obtained from parallel pump and probe polarization geometries (ΔA_‖) are plotted in green circles, and the spectra from perpendicular polarization geometries (ΔA_⊥) are plotted in dark-red triangles. A–C, Inset, shows the changes in apparent anisotropy of the ¹³C¹⁵N attached to the sole thiol of the indicated mutant form of KSI, at different times after the photoexcitation of the enzyme-bound C183, in the ground-state bleach (blue triangles) and the electronic excited state (dark-red circles). The anisotropy was calculated using Eq. 2 with ΔA_‖ and ΔA_⊥ signals at (A) 2,088.3, (B) 2,090.1, and (C) 2,088.1 cm^-1, respectively, for the electronic ground state and at (A) 2,083.0, (B) 2,085.7, and (C) 2,083.7 cm^-1, respectively, for the electronic excited state. The solid blue and red line through the data in the insets is a fit to a straight line equation.