Electrostatic Fields near the Active Site of Human Aldose Reductase: 2. New Inhibitors and Complications Caused by Hydrogen Bonds

Abstract

Vibrational Stark effect spectroscopy was used to measure electrostatic fields in the hydrophobic region of the active site of human aldose reductase (hALR2). A new nitrile-containing inhibitor was designed and synthesized, and the x-ray structure of its complex, along with cofactor NADP+, with wild-type hALR2 was determined at 1.3 Å resolution. The nitrile is found to be in close proximity to T113, consistent with a hydrogen bond interaction. Two vibrational absorption peaks were observed at room temperature in the nitrile region when the inhibitor binds to wild-type hALR2, indicating that the nitrile probe experiences two different microenvironments, and these could be empirically separated into a hydrogen bonded and non-hydrogen bonded population by comparison with the mutant T113A, where a hydrogen bond to the nitrile is not present. Classical molecular dynamics simulations based on the structure predict a double-peaked distribution in protein electric fields projected along the nitrile probe. The interpretation of these two peaks as a hydrogen bond formation-dissociation process between the probe nitrile group and a nearby amino acid side chain is used to explain the observation of two IR bands, and the simulations were used to investigate the molecular details of this conformational change. Hydrogen bonding complicates the simplest analysis of vibrational frequency shifts as being due solely to electrostatic interactions through the vibrational Stark effect, and the consequences of this complication are discussed.

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

“…The quantitative agreement between experiment and computation for M116C-CN and M105C-CN contrasts with prior studies of electrostatic changes in proteins due to pH changes, side-chain mutation, and ligand binding, in which qualitative agreement, at best, has been observed between experiment and theory (20,44,45,57,58). We hypothesize that our simple computational approach succeeded in the present study due to the subtle perturbations of the homologous series of bound phenols (differing only in their meta and para substituent groups) and to the absence of structural and solvent rearrangements that are likely to accompany more gross perturbations, such as pH changes, mutation, or protein-ligand association.…”

Molecular quantum mechanical gradients within the polarizable embedding approach—Application to the internal vibrational Stark shift of acetophenone

“…Vibrational probes have proven to be particularly well suited to act as local and directional metrics of protein electric fields via the vibrational Stark effect, [9][10][11][12][13][14][15][16][17] which describes the susceptibility of a vibrational transition to an electric field. Often one uses "internal" or "external" to distinguish the source of the Stark effect, being either a surrounding environment or an externally applied field.…”

Modeling biophysical and biological properties from the characteristics of the molecular electron density, electron localization and delocalization matrices, and the electrostatic potential

“…Modern ultrahigh resolution X‐ray crystallography is capable of delivering accurate experimental MESPs and electric fields even within the confines of an enzyme active site. Probing the electric field inside an active site is of paramount importance in understanding enzyme catalysis as has been emphasized in the recent literature …”

“…The intensity of the electric field in this enzyme active site is reflected in the density of the field lines and averages to a magnitude of the order of 10 9 V m −1 near the centroid of the cavity (B. Guillot, A. Podjarny, Private communication 2013). These electric field strengths, typically encountered in enzyme active sites, can alter the rates of chemical reactions significantly and can have detectable IR vibrational Stark shifts …”

“…Through the use of calibration curves, vibrational Stark shifts exhibited by small host molecules or chromophoric groups in molecules trapped into enzyme active sites can be used to probe the strengths and directions of the local electric fields. Such studies have been carried out for a number of host–protein systems: CO bound to the Fe of the porphyrin of myoglobin, and CN (nitrile)‐containing substrates complexed at the human aldose reductase enzyme (hALR2) active site are two notable examples. The changes in the local fields accompanying point mutations in the active site have been monitored through the observation of the corresponding vibrational Stark shifts of the nitrile group IR fingerprint by Boxer et al (See also Refs.…”