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

Abstract

Hydrogen bond networks are key elements of protein structure and function but have been challenging to study within the complex protein environment. We have carried out in-depth interrogations of the proton transfer equilibrium within a hydrogen bond network formed to bound phenols in the active site of ketosteroid isomerase. We systematically varied the proton affinity of the phenol using differing electron-withdrawing substituents and incorporated sitespecific NMR and IR probes to quantitatively map the proton and charge rearrangements within the network that accompany incremental increases in phenol proton affinity. The observed ionization changes were accurately described by a simple equilibrium proton transfer model that strongly suggests the intrinsic proton affinity of one of the Tyr residues in the network, Tyr16, does not remain constant but rather systematically increases due to weakening of the phenol-Tyr16 anion hydrogen bond with increasing phenol proton affinity. Using vibrational Stark spectroscopy, we quantified the electrostatic field changes within the surrounding active site that accompany these rearrangements within the network. We were able to model these changes accurately using continuum electrostatic calculations, suggesting a high degree of conformational restriction within the protein matrix. Our study affords direct insight into the physical and energetic properties of a hydrogen bond network within a protein interior and provides an example of a highly controlled system with minimal conformational rearrangements in which the observed physical changes can be accurately modeled by theoretical calculations.computational modeling | enzyme catalysis | protein electrostatics | protein semisynthesis | active site environment H ydrogen bond networks are ubiquitous structural features within proteins, and they play key roles linking secondary and tertiary structural elements and spanning protein-protein interfaces. Such networks are especially common within enzyme active sites, where they position protein and substrate groups for catalysis, stabilize charge rearrangements during chemical transformations, and mediate proton transfers (1). Despite the prevalence and critical structural and functional roles of hydrogen bond networks, incisive dissection of their physical properties within the idiosyncratic interior of folded proteins remains difficult.Hydrogen-bonded protons are not observed in the vast majority of protein X-ray structures due to the low X-ray scattering power of hydrogen atoms (2). Thus, the presence of hydrogen bond networks is typically inferred from the proximity and orientation of hydrogen bond donor and acceptor groups within refined protein structural models. The inherent inability of most X-ray diffraction studies to monitor proton positions imposes additional challenges for dissecting the physical features that influence the equilibrium protonation states of specific residues along a hydrogen-bonded proton transfer network. Furthermore, it remains extremely challenging t...

Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

“…1C) (16). In several high-resolution crystal structures, these distances are found to be around 2.6 Å (14,16,17), which is much shorter than those observed in hydrogen-bonded liquids such as water, where O-O distances are typically around 2.85 Å. Such short heavy-atom distances are only slightly larger than those typically associated with low-barrier hydrogen bonds (18)(19)(20), where extensive proton sharing is expected to occur between the atoms.…”

“…S4) (14). This analysis yielded values of 79% for the Tyr57 ionization for H and 86% for D (Table S3) compared with simulated values of 94.2% and 98.3% (±0.3%), respectively.…”

“…Recent experiments have investigated how the binding of intermediate analogs to KSI D40N affects the sharing of ionization along the extended hydrogen-bond network that is formed (Fig. 1B) (14). These experiments identified that ionization sharing is maximized when phenol, whose solution pK a of 10 equals that of the actual intermediate of KSI (Fig.…”

“…These experiments identified that ionization sharing is maximized when phenol, whose solution pK a of 10 equals that of the actual intermediate of KSI (Fig. 1A), is bound (14,41,42). We thus performed AI-PIMD simulations of the KSI D40N · phenol complex.…”

“…1B), therefore permitting experimental investigation of an intermediate-like state of the enzyme (6,(12)(13)(14). Experiments have identified that, in the absence of an inhibitor, one of the residues in the active site of KSI D40N is deprotonated (12).…”