Dissecting Proton Delocalization in an Enzyme’s Hydrogen Bond Network with Unnatural Amino Acids

Wu, Yufan; Fried, Stephen D.; Boxer, Steven G.

doi:10.1021/acs.biochem.5b00958

Cited by 20 publications

(47 citation statements)

References 25 publications

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“…Similar to the effect on the catalysis of the slow substrate 5(10)-estrene-3,17-dione (Table S1) 10 , small but systematic changes were observed in these conservative variants, making them ideal candidates to critically test the electric field/activation free energy correlations mapped by vibrational Stark spectroscopy. 3 …”

Section: Resultsmentioning

confidence: 74%

“…More specifically in the case of KSI (and a number of other enzymes), the removal of specific H-bond interactions prevents a direct comparison of electrostatic stabilization with a widely-discussed alternative proposal that KSI’s active site preferentially forms a strong, short H-bond with the intermediate relative to the substrate to provide additional stabilization energy. 7,8 Therefore, in this work we introduce more subtle and conservative changes by site-specifically replacing each of the key Tyr residues with 3-Cl-Tyr (Cl-Y) 9 in the KSI active site 10 to provide a critical test of the earlier analysis on electric field/activation free energy correlations. We then probe the physical basis of the large electric field and demonstrate the functional connection between the preferential H-bond interactions and electrostatic stabilization.…”

Section: Introductionmentioning

confidence: 99%

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A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase

2016

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The vibrational Stark effect (VSE) has been used to measure the electric field in the active site of ketosteroid isomerase (KSI). These measured fields correlate with ΔG‡ in a series of conventional mutants yielding an estimate for the electrostatic contribution to catalysis (Fried et al. Science, 2014, 346, 1510–1513). In this work we test this result with much more conservative variants in which individual Tyr residues in the active site are replaced by 3-chlorotyrosine via amber suppression. The electric fields sensed at the position of the carbonyl bond involved in charge displacement during catalysis were characterized using the VSE, where the field sensitivity has been calibrated by vibrational Stark spectroscopy, solvatochromism, and MD simulations. A linear relationship is observed between the electric field and ΔG‡ that interpolates between wild-type and more drastic conventional mutations, reinforcing the evaluation of the electrostatic contribution to catalysis in KSI. A simplified model and calculation are developed to estimate changes in the electric field accompanying changes in the extended hydrogen-bond network in the active site. The results are consistent with a model in which the O-H group of a key active site tyrosine functions by imposing a static electrostatic potential onto the carbonyl bond. The model suggests that the contribution to catalysis from the active site hydrogen bonds is of similar weight to the distal interactions from the rest of the protein. A similar linear correlation was also observed between the proton affinity of KSI’s active site and the catalytic rate, suggesting a direct connection between the strength of the H-bond and the electric field it exerts.

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Section: Resultsmentioning

confidence: 74%

Section: Introductionmentioning

confidence: 99%

A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase

2016

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“…If the asymmetrically labeled F n Y 32 side chains have multiple rotameric states, 18,49 it has little influence on their E °’s.…”

Section: Discussionmentioning

confidence: 99%

Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer

Ravichandran¹,

Zong

Taguchi³

et al. 2017

J. Am. Chem. Soc.

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Redox-active tyrosines (Ys) play essential roles in enzymes involved in primary metabolism including energy transduction and deoxynucleotide production catalyzed by ribonucleotide reductases (RNRs). Thermodynamic characterization of Ys in solution and in proteins remains a challenge due to the high reduction potentials involved and the reactive nature of the radical state. The structurally characterized α3Y model protein has allowed the first determination of formal reduction potentials (E°′) for a Y residing within a protein (Berry, B. W.; Martínez-Rivera, M. C.; Tommos, C. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 9739–9743). Using Schultz’s technology, a series of fluorotyrosines (FnY, n = 2 or 3) was site-specifically incorporated into α3Y. The global protein properties of the resulting α3(3,5)F2Y, α3(2,3,5)F3Y, α3(2,3)F2Y and α3(2,3,6)F3Y variants are essentially identical to those of α3Y. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and E°’s of the very high-potential α3FnY proteins. E°′(pH 5.5; α3FnY(O•/OH)) spans a range of 1040 ± 3 mV to 1200 ± 3 mV versus the normal hydrogen electrode. This is comparable to the potentials of the most oxidizing redox cofactors in Nature. The FnY analogs, and the ability to site-specifically incorporate them into any protein of interest, provide new tools for mechanistic studies on redox-active Ys in proteins and on functional and aberrant hole-transfer reactions in metallo-enzymes. The former application is illustrated here by using the determined α3FnY ΔE°’s to model the thermodynamics of radical-transfer reactions in FnY-RNRs and to experimentally test and support the key prediction made.

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“…As shown in Table I, in Cl-Tyr16 KSI D40N f 16 /f 57 is increased by 1.34-fold compared to the unlabeled protein, which is in excellent agreement with the experimentally measured value of 1.45-fold. 33 In contrast, chlorinating Tyr57 reduces proton sharing in the triad, which decreases f 16 /f 57 by 1.2 fold compared to the unlabeled case.…”

Section: A Change In Partial Ionizations In Response To Mutationsmentioning

confidence: 99%

“…2a and 2b, respectively. In aqueous solution, chlorination lowers the side-chain pK a of tyrosine by 1.8 units, 33 which is equivalent to stabilizing the deprotonated state by 2.5 kcal/mol. If one were to assume a simple two-state model to describe the proton transfer between Tyr16 and Tyr57, i.e., H16 is attached to either one tyrosine or the other (Fig.…”

Section: A Change In Partial Ionizations In Response To Mutationsmentioning

confidence: 99%

Proton Network Flexibility Enables Robustness and Large Electric Fields in the Ketosteroid Isomerase Active Site

2017

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Hydrogen-bond networks play vital roles in biological functions ranging from protein folding to enzyme catalysis. Here we combine electronic structure calculations and ab initio path integral molecular dynamics simulations, which incorporate both nuclear and electronic quantum effects, to show why the network of short hydrogen bonds in the active site of ketosteroid isomerase is remarkably robust to mutations along the network and how this gives rise to large local electric fields. We demonstrate that these properties arise from the network's ability to respond to a perturbation by shifting proton positions and redistributing electronic charge density. This flexibility leads to small changes in properties such as the partial ionization of residues and pK isotope effects upon mutation of the residues, consistent with recent experiments. This proton flexibility is further enhanced when an extended hydrogen-bond network forms in the presence of an intermediate analogue, which allows us to explain the chemical origins of the large electric fields in the enzyme's active site observed in recent experiments.

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Dissecting Proton Delocalization in an Enzyme’s Hydrogen Bond Network with Unnatural Amino Acids

Cited by 20 publications

References 25 publications

A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase

A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase

Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer

Proton Network Flexibility Enables Robustness and Large Electric Fields in the Ketosteroid Isomerase Active Site

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