Detection of Large pK Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes

O ver the past decades, visualization of enzyme threedimensional structures has revealed that active sites are located in crevices or pockets. Within these active sites are found coenzymes, cofactors, metal ions, and side chains that participate in a rich array of chemical reactions. Based on this information and decades of enzymology and bioorganic chemistry, reasonable chemical mechanisms using these groups can be posited for most enzymatic reactions. Nevertheless, we still cannot quantitatively account for the enormous enzymatic rate enhancements and exquisite specificities observed by enzymes.Over the past century, Polanyi, Haldane, Pauling, and Jencks recognized a key distinguishing feature between chemical reactions taking place in an enzyme's active site and the corresponding reaction taking place in solution (1-4). Even if the enzymatic and solution reactions use the same cofactors, metal ions, and functional groups, an enzyme can use noncovalent interactions between the enzyme and substrate to accelerate the reaction. Indeed, binding interactions between enzymes and substrates, both directly at the site of chemical transformation and with nonreacting portions of the substrate have been shown to contribute to catalysis (e.g., refs. 5-19). The mechanisms by which these noncovalent interactions can assist catalysis have been widely discussed, as briefly described in the following two paragraphs.In the simplest scenario an enzyme can use binding interactions to localize the substrate to the active site. Beyond simple localization, these binding interactions can correctly position the reactive portion of a substrate relative to active site functional groups and relative to other substrates (7, 20-25). Concomitant with substrate binding solvent is displaced and excluded from the active site and solvent exclusion may be important in shaping the electrostatic environment within the active site (26,27). Indeed, solvent exclusion by substrate binding has been suggested to be important for catalysis in numerous enzymes (e.g., refs. 26-32).Jencks and others realized that remote binding interactions can do more than provide for tight binding between substrate and enzyme. Reactions of bound substrates can be facilitated by use of so-called ''intrinsic binding energy'', which can pay for substrate desolvation, distortion, electrostatic destabilization, and entropy loss (3,7,33,34). The term intrinsic binding energy is not a molecular explanation for catalysis, but rather provides a conceptual framework for analyzing the energetics of enzymatic catalysis. In this scenario the maximum binding energy is not realized in the ground state, because aspects of the bound state, such as restricted positioning of substrates, are energetically unfavorable relative to the interactions and freedom of motion in aqueous solution. However, changes associated with achievement of the transition state, such as charge and geometric rearrangements and the formation of partial covalent bonds between positioned substrates, allow the binding ...

Section: Discussioncontrasting

confidence: 50%

mentioning

confidence: 99%

Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase

Schwans

Kraut

Herschlag

2009

“…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.…”

mentioning

confidence: 83%

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confidence: 95%

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

Wang

Fried

Boxer

et al. 2014

136

178

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.enzyme | hydrogen bonding | nuclear quantum effects | proton delocalization | ab initio path integral molecular dynamics A lthough 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 groundstate 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.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)(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). Although one might expect the carboxylic acid of Asp103 to be deprotonated, the combination of recent 13 C NMR and ultraviolet visible spectroscopy (UVVis) e...

“…These compounds have estimated pK a values of 15.0 (5-Andro) and 14.4 (4-Andro), and 5-Andro was previously shown to bind pKSI D40N in a structurally similar manner to phenols, with the hydroxyl oxygen of its cyclohexanolic A-ring positioned within hydrogen bonding distance of Y16 and D103 (28). Furthermore, prior studies have shown that the multiple distal rings of a steroid do not alter the electrostatic environment within the oxyanion hole, relative to a bound single-ring phenol (29).…”

Section: Nmr Identification Of Tyr Ionizations In Ksi-phenol Complexementioning

confidence: 99%

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

Sigala¹,

Fafarman²,

Schwans³

et al. 2013

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...