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

Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds.Although many biological processes can be well-described with classical mechanics, there has been much interest and debate as to the role of quantum effects in biological systems ranging from photosynthetic energy transfer, to photoinduced isomerization in the vision cycle and avian magnetoreception (1). For example, nuclear quantum effects, such as tunneling and zero-point energy (ZPE), have been observed to lead to kinetic isotope effects of greater than 100 in biological proton and proton-coupled electron transfer processes (2, 3). However, the role of nuclear quantum effects in determining the ground-state thermodynamic properties of biological systems, which manifest as equilibrium isotope effects, has gained significantly less attention (4).Ketosteroid isomerase (KSI) possesses one of the highest enzyme unimolecular rate constants and thus, is considered a paradigm of proton transfer catalysis in enzymology (5–11). The remarkable rate of KSI is intimately connected to the formation of a hydrogen-bond network in its active site (Fig. 1A), which acts to stabilize a charged dienolate intermediate, lowering its free energy by ∼11 kcal/mol (1 kcal = 4.18 kJ) relative to solution (Fig. S1) (6). This extended hydrogen-bond network in the active site links the substrate to Asp103 and Tyr16, with the latter further hydrogen-bonded to Tyr57 and Tyr32, which is shown in Fig. 1A.Open in a separate windowFig. 1.KSI⋅intermediate and KSI^D40N ? inhibitor complex. Schematic depiction of (A) the KSI⋅intermediate complex during the catalytic cycle (Fig. S1) and (B) a complex between KSI^D40N and phenol, an inhibitor that acts as an intermediate analog. Both the intermediate and the inhibitor are stabilized by a hydrogen-bond network in the active site of KSI. (C) Image of KSI^D40N with the tyrosine triad enlarged and the atoms O16, H16, O32, H32, and O57 labeled (shown with Tyr57 deprotonated) (16).The mutant KSI^D40N preserves the structure of the wild-type (WT) enzyme while mimicking the protonation state of residue 40 in the intermediate complex (Fig. 1B), therefore permitting experimental investigation of an intermediate-like state of the enzyme (6, 12–14). Experiments have identified that, in the absence of an inhibitor, one of the residues in the active site of KSI^D40N is deprotonated (12). Although one might expect the carboxylic acid of Asp103 to be deprotonated, the combination of recent ¹³C NMR and ultraviolet visible spectroscopy (UV-Vis) experiments has shown that the ionization resides primarily on the hydroxyl group of Tyr57, which possesses an anomalously low pK_a of 6.3 ± 0.1 (12). Such a large tyrosine acidity is often associated with specific stabilizing electrostatic interactions (such as a metal ion or cationic residue in close proximity), which is not the case here, suggesting that an additional stabilization mechanism is at play (15).One possible explanation is suggested by the close proximity of the oxygen (O) atoms on the side chains of the adjacent residues Tyr16 (O16) and Tyr32 (O32) to the deprotonated O on Tyr57 (O57) (Fig. 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 (18–20), where extensive proton sharing is expected to occur between the atoms. In addition, at these short distances, the proton’s position uncertainty (de Broglie wavelength) becomes comparable with the O–O distance, indicating that nuclear quantum effects could play an important role in stabilizing the deprotonated residue (Fig. 1C). In this work, we show how nuclear quantum effects determine the properties of protons in the active-site hydrogen-bond network of KSI^D40N in the absence and presence of an intermediate analog by combining ab initio path integral simulations and isotope effect experiments.