Vibrational Stark Effects of Carbonyl Probes Applied to Reinterpret IR and Raman Data for Enzyme Inhibitors in Terms of Electric Fields at the Active Site

Abstract

IR and Raman frequency shifts have been reported for numerous probes of enzyme transition states, leading to diverse interpretations. In the case of the model enzyme ketosteroid isomerase (KSI), we have argued that IR spectral shifts for a carbonyl probe at the active site can provide a connection between the active site electric field and the activation free energy (Fried et al. Science, 2014, 346, 1510–1514). Here we generalize this approach to a much broader set of carbonyl probes (e.g. oxoesters, thioesters, and amides), first establishing the sensitivity of each probe to an electric field using vibrational Stark spectroscopy, vibrational solvatochromism, and MD simulations, and then applying these results to re-interpret data already in the literature for enzymes such as 4-chlorobenzoyl-CoA dehalogenase and serine proteases. These results demonstrate that the vibrational Stark effect provides a general framework for estimating the electrostatic contribution to the catalytic rate and may provide a metric for the design or modification of enzymes. Opportunities and limitations of the approach are also described.

Solvent-Independent Anharmonicity for Carbonyl Oscillators

“…30,58,63,64 This expands upon prior work, which has characterized the Stark tuning rate of various carbonyl probes, finding that the Stark tuning rate is constant across multiple solvents both with and without H-bonding, allowing for quantitative mapping of electric fields from observed vibrational frequencies. 7,8 That is, solvents exerting different electric fields do not perturb the bond’s force constant or atomic charges, as described by bond polarization models, but rather modify the corresponding energy levels according to the VSE. This has important ramifications for interpretation and rationalization of carbonyl frequency shifts in the condensed phase.…”

“…It will be very interesting to extend these measurements to carbonyl probes at the active sites of enzymes where even larger frequency shifts to the red, interpreted as still larger electric fields than those in solution, are present. 8,9,16 …”

“…This measurement is common in the gas phase by measuring overtone and combination bands but less common in condensed phases largely because these transitions are weak and often overwhelmed by modes from the solvent. 30 While the vibrational Stark spectrum of acetophenone and other carbonyl probes in a mixed frozen solvent where both H-bonded and non-H-bonded forms are present can be fit with a single $false|\mathrm{normal\Delta }{\overrightarrow{\mu }}_{\mathrm{normalC}=\mathrm{normalO}}false|f$, 7,8 2D IR can provide a more direct measurement of the anharmonicity in fluid solution because both the ν = 0 → 1 $false({\overline{\nu }}_{01}false)$ diagonal peak and the ν = 1 → 2 $false({\overline{\nu }}_{12}false)$ off-diagonal peak are readily observed. The anharmonic shift, Δ, is given by subtracting the frequencies of the ν = 0 → 1 and ν = 1 → 2 peaks, for example, $\mathrm{normal\Delta }={\overline{\nu }}_{01}-{\overline{\nu }}_{12}$.…”

Electric Fields and Enzyme Catalysis

“…In contrast, data from our laboratory accrued over the past few years have supported the view that most vibrational frequency variation (in carbonyls, at least) can be explained through a static electric field–difference dipole effect (53, 91), not by bond weakening or strain (i.e., changes in the force constant) (103, 104). This represents a departure from how vibrational measurements (both infrared and Raman) have traditionally been interpreted in enzymology (105–107), and efforts are ongoing to reinterpret this corpus of biochemical data within this physical framework (108, 109). …”

“…The field-barrier plot (Figure 5 b ) analogous to that drawn up for KSI (Figure 4 d ) will therefore not capture the full catalytic effect implied by the more general Equation 1 (108). Hence, experiments are needed that measure the projection of the active site electric field on both the transition state ( $\mid {\overrightarrow{F}}_{\text{enz},\text{TS}}^{\mathrm{C}=\mathrm{O}}\mid$) and ground state ( $\mid {\overrightarrow{F}}_{\text{enz},\text{AE}}^{\mathrm{C}=\mathrm{O}}\mid$) geometries to assign values to both terms in the equation.…”