NMR Localization of Protons in Critical Enzyme Hydrogen Bonds

Sharif, Shasad; Fogle, Emily J.; Toney, Michael D.; Денисов, Г. С.; Shenderovich, Ilya G.; Buntkowsky, Gerd; Tolstoy, Peter M.; Huot, Monique Chan; Limbach, Hans−Heinrich

doi:10.1021/ja0728223

Cited by 69 publications

(81 citation statements)

References 22 publications

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“…39,40 For example, early studies of aspartate aminotransferase confirmed that some enzymes use the cofactor with a fully protonated PN, which then forms a salt-bridge with Asp222 below the pyridine ring. 41 However, recent studies suggest that some PLP enzymes, such as alanine racemase and O-acetylserine sulfhydrylase, use the PN in an unprotonated form. This remodulation of the PLP structure demonstrates that not all PLP enzymes require the protonated PN for stabilization of carbanionic intermediates.…”

Section: Resultsmentioning

confidence: 99%

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

Huang

Kang

Chang

2014

J of Molecular Recognition

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The importance of protonation states and proton transfer in pyridoxal 5′-phosphate (PLP)-chemistry can hardly be overstated. Although experimental approaches to investigate pKa values can provide general guidance for assigning proton locations, only static pictures of the chemical species are available. To obtain the overall protein dynamics for the interpretation of detailed enzyme catalysis in this study, guided by information from solid-state NMR, we performed molecular dynamics (MD) simulations for the PLP-dependent enzyme tryptophan synthase (TRPS), whose catalytic mechanism features multiple quasi-stable intermediates. The primary objective of this work is to elucidate how the position of a single proton on the reacting substrate affects local and global protein dynamics during the catalytic cycle. In general, proteins create a chemical environment and an ensemble of conformational motions to recognize different substrates with different protonations. The study of these interactions in TRPS shows that functional groups on the reacting substrate, such as the phosphoryl group, pyridine nitrogen, phenolic oxygen and carboxyl group, of each PLP-bound intermediate play a crucial role in constructing an appropriate molecular interface with TRPS. In particular, the protonation states of the ionizable groups on the PLP cofactor may enhance or weaken the attractions between the enzyme and substrate. In addition, remodulation of the charge distribution for the intermediates may help generate a suitable environment for chemical reactions. The results of our study enhance knowledge of protonation states for several PLP intermediates and help to elucidate their effects on protein dynamics in the function of TRPS and other PLP-dependent enzymes.

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Section: Resultsmentioning

confidence: 99%

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

Huang

Kang

Chang

2014

J of Molecular Recognition

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“…8,35−41 In particular, 15 N SSNMR chemical shift measurements of the pyridine ring nitrogen in the internal aldimine form of aspartate aminotransferase confirm that at the start of catalysis the pyridine nitrogen is protonated and ready to assist in the formation of a canonical quinonoid intermediate. 8 This is in contrast to the neutral pyridine nitrogen found by SSNMR in the resting internal aldimine forms of tryptophan synthase and alanine racemase.…”

Section: Introductionmentioning

confidence: 93%

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

et al. 2016

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Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5′-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography—the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry—to interrogate a carbanionic/quinonoid intermediate analogue in the β-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4′ of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for β-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

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“…Whereas for NHN and OHN hydrogen bonds 15 N substitution provides interesting NMR hydrogen bond correlations 13−16 which have been applied to biomolecules, 17,18 it is much more difficult to obtain information about the geometries of OHO by NMR because of the absence of a nucleus with spin 1 / 2 . Here, only the 1 H chemical shift of the bridging proton, δ( 1 H), has been correlated in the past with the O···O, O···H, and H···O distances for OHO type hydrogen bonds.…”

Section: ■ Introductionmentioning

confidence: 99%

Hydrogen Bond Geometries and Proton Tautomerism of Homoconjugated Anions of Carboxylic Acids Studied via H/D Isotope Effects on ¹³C NMR Chemical Shifts

et al. 2012

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Ten formally symmetric anionic OHO hydrogen bonded complexes, modeling Asp/Glu amino acid side chain interactions in nonaqueous environment (CDF(3)/CDF(2)Cl solution, 200-110 K) have been studied by (1)H, (2)H, and (13)C NMR spectroscopy, i.e. intermolecularly H-bonded homoconjugated anions of acetic, chloroacetic, dichloroacetic, trifluoroacetic, trimethylacetic, and isobutyric acids, and intramolecularly H-bonded hydrogen succinate, hydrogen rac-dimethylsuccinate, hydrogen maleate, and hydrogen phthalate. In particular, primary H/D isotope effects on the hydrogen bond proton signals as well as secondary H/D isotope effects on the (13)C signals of the carboxylic groups are reported and analyzed. We demonstrate that in most of the studied systems there is a degenerate proton tautomerism between O-H···O(-) and O(-)···H-O structures which is fast in the NMR time scale. The stronger is the proton donating ability of the acid, the shorter and more symmetric are the H-bonds in each tautomer of the homoconjugate. For the maleate and phthalate anions exhibiting intramolecular hydrogen bonds, evidence for symmetric single well potentials is obtained. We propose a correlation between H/D isotope effects on carboxylic carbon chemical shifts and the proton transfer coordinate, q(1) = ½(r(OH) - r(HO)), which allows us to estimate the desired OHO hydrogen bond geometries from the observed (13)C NMR parameters, taking into account the degenerate proton tautomerism.

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NMR Localization of Protons in Critical Enzyme Hydrogen Bonds

Cited by 69 publications

References 22 publications

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

Hydrogen Bond Geometries and Proton Tautomerism of Homoconjugated Anions of Carboxylic Acids Studied via H/D Isotope Effects on ¹³C NMR Chemical Shifts

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NMR Localization of Protons in Critical Enzyme Hydrogen Bonds

Cited by 69 publications

References 22 publications

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

Switches of hydrogen bonds during ligand–protein association processes determine binding kinetics

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity

Hydrogen Bond Geometries and Proton Tautomerism of Homoconjugated Anions of Carboxylic Acids Studied via H/D Isotope Effects on 13C NMR Chemical Shifts

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Hydrogen Bond Geometries and Proton Tautomerism of Homoconjugated Anions of Carboxylic Acids Studied via H/D Isotope Effects on ¹³C NMR Chemical Shifts