Solution Structure of 3-Oxo-Δ 5 -Steroid Isomerase

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: Comparison Of Ksi Catalysis Of Reactions Of the Single-ring mentioning

confidence: 55%

“…the multiple rings prevent water from approaching within Ϸ10 Å of the oxyanion hole in the enzyme-substrate complex (40,41). Remote substrate binding interactions in KSI could position the substrate relative to the oxyanion hole and the general acid/base and could also influence the electrostatic environment by excluding solvent from the enzyme active site.…”

mentioning

confidence: 99%

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

Schwans

Kraut

Herschlag

2009

“…A priori, the carboxylic acid moiety of an aspartic acid (solution pK a of ∼4) would be expected to be much more acidic than that of a Tyr hydroxyl group (solution pK a of ∼10) (24). However, D103 is surrounded by hydrophobic residues that elevate its pK a well above its typical solution value (15,25,26), and recent quantum mechanics/molecular mechanics (QM/MM) studies of phenols bound to pKSI D40N have suggested that D103 may be less acidic than Y16 (11). Furthermore, we observed that the D103N/D40N mutant, which preserves hydrogen bonding to residue 103 but ablates its ability to transfer a proton, still binds 4-F-3-Me-phenol (pK a = 9.8) predominantly in its neutral form (Fig.…”

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...

“…Rather, the ring edge of a Phe residue, Phe54 for one oxygen and Phe116 for the other oxygen, is situated next to these anionic oxygen atoms (Fig. 2B) (15,16). We posited, based on this structural information, that anion-aromatic interactions between the anionic Asp and Phe54 and Phe116 were used instead of a hydrogen-bond network to position the general base in the KSI active site and facilitate catalysis.…”

mentioning

confidence: 99%

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Schwans¹,

Sunden²,

Lassila³

et al. 2013

Although the cation-pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has been shown to be important in protein structure and protein-ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion-aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion-aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogenbonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion-aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.enzyme catalysis | general-base catalysis | noncovalent interactions E nzymes use the same functional groups to achieve "chemical catalysis" as small-molecule catalysts, and yet enzymes attain much greater rate enhancements. A distinguishing feature of enzymes is that their reactions occur in highly specialized active sites that use noncovalent interactions to precisely position enzymatic functional groups relative to substrates and relative to other active-site features. Understanding how these groups are positioned within enzyme active sites is key for understanding the differences between enzymes and small-molecule catalysts.It is generally recognized that hydrophobic interactions can help bind and position substrates in enzyme active sites (1, 2). Beyond hydrophobic interactions, much attention has focused on the importance of hydrogen bonds in precisely positioning active-site groups directly involved in the chemical reaction (e.g., the catalytic triad in serine proteases), and the importance of hydrogen-bonding groups is supported by mutagenesis in many cases (e.g., refs. 1 and 3-5).In addition to hydrogen bonds, the cation-pi pair, formed between a side-chain or substrate cation and the negative electrostatic potential associated with the face of a pi system, has been widely discussed in hundreds of literature reports and has been shown to be important in both protein structure and proteinligand interactions (e.g., refs. 6 and 7). There has been much ...