Proton Affinity of the Oxyanion Hole in the Active Site of Ketosteroid Isomerase

Childs, W. V.; Boxer, Steven G.

doi:10.1021/bi100074s

Cited by 29 publications

(73 citation statements)

References 36 publications

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“…This series of spectra strongly suggests that single-ring phenols bind to the pKSI D40N mutant with a neutral phenol fraction that becomes significant at a phenol pK a greater than 8 and increases with the phenol pK a until the protonated phenol is the predominantly bound form at or above a pK a of 10. This conclusion is consistent with prior UV and IR studies (13,22), and it is further supported by 13 C NMR and IR results described below.…”

Section: Resultssupporting

confidence: 92%

“…Recent spectroscopic studies suggested that single-and multiple-ring phenolic ligands are bound to pKSI D40N with a neutral, protonated fraction that increases with ligand pK a and reaches a ratio of 50:50 ionized/neutral for a ligand with a solution pK a of 9.7 (13,22). To dissect and understand the nature and properties of proton transfer within the KSI oxyanion hole, we turned to systematic studies with single-ring phenols, because these compounds are available over a wider pK a range than naphthols or steroids.…”

Section: Resultsmentioning

confidence: 99%

“…1A), have provided evidence that ligands of increasing pK a are bound as an increasing population of neutral, protonated phenol (11,13,22). These results suggest that an unspecified active site residue can ionize with increasing phenol pK a , resulting in a net proton transfer to the bound ligand.…”

Section: Significancementioning

confidence: 93%

“…The chemical shift of the C ζ carbon of Tyr, the carbon atom adjacent to the hydroxyl group, is highly sensitive to the ionization state of Tyr, shifting down-field from 155.5 to 166.3 ppm upon ionization in Tyr-containing peptides in aqueous solution (24). We therefore used 13 C NMR of phenol complexes of pKSI D40N containing 13 C ζ -labeled Tyr residues to determine whether Y16 or any of the active site Tyr residues ionize upon phenol binding.…”

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

confidence: 99%

“…Bacterial ketosteroid isomerase (KSI) from Pseudomonas putida KSI (pKSI) and Comamonas testosteroni KSI has been a powerful system with which to study the physical properties of hydrogen bonds within an enzyme active site (6)(7)(8)(9)(10)(11)(12)(13). KSI uses a general base, D40 (pKSI numbering), to deprotonate steroid substrates and form a dienolate reaction intermediate that is stabilized by hydrogen bonds donated by Y16 and protonated D103.…”

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confidence: 99%

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

Sigala¹,

Fafarman²,

Schwans³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 93%

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

confidence: 99%

mentioning

confidence: 99%

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

Sigala¹,

Fafarman²,

Schwans³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Enantioselective Cyanoethoxycarbonylation of Isatins Promoted by a Lewis Base–Brønsted Acid Cooperative Catalyst

Ogura¹,

Akakura²,

Sakakura³

et al. 2013

Angewandte Chemie

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Enantioselective Cyanoethoxycarbonylation of Isatins Promoted by a Lewis Base–Brønsted Acid Cooperative Catalyst

et al. 2013

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Oxindole is an important core structure found in many natural and synthetic bioactive compounds. [1] For the chemical synthesis of these useful bioactive compounds, much attention has been devoted to the development of stereoselective carbon-carbon bond-forming reactions at the C3 carbonyl carbon atom of isatins, and many enantioselective methods have been reported. [2] However, the enantioselective cyanation of isatins has not yet been reported. Enantioselective cyanation affords the corresponding cyanohydrin or its equivalent, which would be a useful chiral building block for the synthesis of these bioactive compounds.The asymmetric cyanation of carbonyl compounds is an important reaction for the construction of tetrasubstituted carbon stereocenters. [3] Representative cyanation methods include hydrocyanation with hydrogen cyanide and silylcyanation with a silyl cyanide. [4] Although many chiral catalysts have been developed for asymmetric hydrocyanation and silylcyanation, these methods require a highly toxic cyanation reagent, and the corresponding cyanation products are rather unstable. In contrast, cyanocarbonylation with a less toxic acyl cyanide or alkyl cyanoformate [5] is also useful for the cyanation of carbonyl compounds, and the products are rather stable.In 2001, Deng and Tian reported the first enantioselective cyanocarbonylation with (DHQ) 2 AQN as a chiral nucleophilic-base catalyst. [6,7] Although this pioneering method is highly efficient for the reaction of ketones, (DHQ) 2 AQN gave poor results in the reaction of N-methylisatin in our study, probably because N-methylisatin is much less reactive than ketones (Scheme 1). We envisioned that acid-base cooperative catalysts, [8] which have a Lewis basic site and a Brønsted acidic site, may be able to promote the enantioselective cyanocarbonylation of isatins. The Lewis basic site would activate the cyanocarbonylation reagent, and the Brønsted acidic site would simultaneously activate the carbonyl group of the isatin through hydrogen bonding to promote the reaction. We report herein the enantioselective cyanoethoxycarbonylation of isatins with acid-base cooperative organocatalysts.On the basis of the findings of the Deng research group and our preliminary experiments, we chose the chiral quinuclidine moiety B1 derived from cinchonidine as the Lewis basic site B in the acid-base cooperative catalyst 1, and optimized the Brønsted acidic site A (Table 1). The reaction of N-methylisatin (R = Me) was conducted with ethyl cyanoformate (1.1 equiv) in CH 2 Cl 2 in the presence of 1 (10 mol %) at ambient temperature. Catalyst 1 a containing sulfonamide A1 as the Brønsted acidic site did not give any products, whereas the use of thiourea A2 gave the desired product in moderate yield with moderate enantioselectivity (Table 1, entries 1 and 2). Upon further investigation of the Brønsted acidic site in catalyst 1, we found that the introduction of a third Brønsted acid (in A3) [9,10] successfully improved both the yield and enantioselectivity to 73 % and 65 % ...

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Proton Affinity of the Oxyanion Hole in the Active Site of Ketosteroid Isomerase

Cited by 29 publications

References 36 publications

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

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

Enantioselective Cyanoethoxycarbonylation of Isatins Promoted by a Lewis Base–Brønsted Acid Cooperative Catalyst

Enantioselective Cyanoethoxycarbonylation of Isatins Promoted by a Lewis Base–Brønsted Acid Cooperative Catalyst

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