Proton Transfer to and from Carbon in Model Reactions

Amyes, Tina L.; Richard, John P.

doi:10.1002/9783527611546.ch30

Cited by 9 publications

(22 citation statements)

References 91 publications

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“…The stock solution of [1- 13 C]-GA in D 2 O was stored at room temperature to minimize the content of glycolaldehyde dimer (19) and the concentration of [1- 13 C]-GA was determined by 1 H NMR spectroscopy as follows. 50 μL of the stock solution of [1- 13 C]-GA in D 2 O was diluted with 700 μL of 30 mM imidazole buffer (20% free base, pD 7.0) in D 2 O and the concentration of [1- 13 C]-GA was determined from comparison of the integrated areas of the signals for the protons of [1- 13 C]-GA hydrate and the C(4,5)-protons of imidazole, with a correction for the presence of 6.1% of the free carbonyl form of GA that is present in equilibrium with the hydrate (19, 29). …”

Section: Methodsmentioning

confidence: 99%

“…The TIM-catalyzed reactions of [1- 13 C]-GA in D 2 O the presence of phosphite dianion were initiated by adding (5 – 20) μL of TIM (ca. 64 units/μL, 12 mg/mL) in 30 mM imidazole buffer (20% free base, pD 7.0) at I = 0.024 in D 2 O to 1.0 mL of a solution containing 20 mM [1- 13 C]-GA, 6 mM imidazole (20% free base, pD 7.0) and 5 - 20 mM phosphite dianion at pD 7.0 and I = 0.1 (NaCl).…”

Section: Methodsmentioning

confidence: 99%

“…The internal standard was the upfield peak of the doublet at 6.7 ppm ( J = 600 Hz) due to the P-H proton of phosphite, or the signal at 7.3 ppm due to the C-(4,5) protons of imidazole.…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme's low molecular weight (dimer, 26 kDa/subunit), high cellular abundance (3), and the centrality of proton transfer at carbon in metabolic processes (4, 5) have made TIM a prominent target for studies on the mechanism of enzyme action (6-8). …”

mentioning

confidence: 99%

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Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-¹³C]-Glycolaldehyde in D₂O

2009

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Product distributions for the reaction of glycolaldehyde labeled with carbon-13 at the carbonyl carbon ([1-13C]-GA) catalyzed by triosephosphate isomerase (TIM) in D2O at pD 7.0 in the presence of phosphite dianion and in its absence were determined by 1H NMR spectroscopy. We observe three products for the relatively fast phosphite-activated reaction (Amyes, T. L., and Richard, J. P. (2007) Biochemistry 46, 5841-5854): [2-13C]-GA from isomerization with intramolecular transfer of hydrogen (12% of products), [2-13C, 2-2H]-GA from isomerization with incorporation of deuterium from D2O at C-2 (64% of the products), and [1-13C, 2-2H]-GA from incorporation of deuterium from D2O at C-2 (23% of products). The much slower unactivated reaction in the absence of phosphite results in formation of the same three products along with the doubly deuterated product [1-13C, 2,2-di-2H]-GA. The two isomerization products ([2-13C]-GA and [2-13C, 2-2H]-GA) are formed in the same relative yields in both the unactivated and the phosphite-activated reactions. However, the additional [1-13C, 2-2H]-GA and the doubly deuterated [1-13C, 2,2-di-2H]-GA formed in the unactivated TIM-catalyzed reaction are proposed to result from a nonspecific reaction(s) at the protein surface. The data provide evidence that phosphite dianion affects the rate, but not the product distribution, of the TIM-catalyzed reaction of [1-13C]-GA at the enzyme active site. They are consistent with the conclusion that both reactions occur at an unstable loop-closed form of TIM, and that activation of the isomerization reaction by phosphite dianion results from utilization of the intrinsic binding energy of phosphite dianion to stabilize the active loop-closed enzyme.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…The internal standard was the upfield peak of the doublet at 6.7 ppm ( J = 600 Hz) due to the P-H proton of phosphite, or the signal at 7.3 ppm due to the C-(4,5) protons of imidazole.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-¹³C]-Glycolaldehyde in D₂O

2009

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“…1000-fold increase in the second-order rate constant for the unactivated isomerization reaction of GA and [1- 13 C]-GA ([( k cat / K m ) E , Scheme 3A] to ( k cat / K m ) E•HPi for catalysis by the enzyme-phosphite complex. 23,50,65 Enzyme activation is rationalized by the model shown in Scheme 3B, where unliganded TIM exists mainly as an inactive open form ( E O ), which is in equilibrium with a small amount of the active closed enzyme ( E C , K C • 1/1000, Scheme 3B). Phosphite dianion, shows a high affinity for binding to E C to form the activated E C •HP i complex [( k cat / K m ) E′ • ( k cat / K m ) E′•HPi , Scheme 3B], with the result that ca .…”

Section: Enzyme Activationmentioning

confidence: 99%

Reflections on the catalytic power of a TIM-barrel

Richard¹,

Zhai²,

Malabanan³

2014

Bioorganic Chemistry

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The TIM-barrel fold is described and its propagation throughout the enzyme universe noted. The functions of the individual front loops of the eponymous TIM-barrel of triosephosphate isomerase are presented in a discussion of: (a) Electrophilic catalysis, by amino acid side chains from loops 1 and 4, of abstraction of an α-carbonyl hydrogen from substrate dihydroxyacetone phosphate (DHAP) or D-glyceraldehyde 3-phosphate (DGAP). (b) The engineering of loop 3 to give the monomeric variant monoTIM and the structure and catalytic properties of this monomer. (c) The interaction between loops 6, 7 and 8 and the phosphodianion of DHAP or DGAP. (d) The mechanism by which a ligand-gated conformational change, dominated by motion of loops 6 and 7, activates TIM for catalysis of deprotonation of DHAP or DGAP. (e) The conformational plasticity of TIM, and the utilization of substrate binding energy to “mold” the distorted active site loops of TIM mutants into catalytically active enzymes. The features of the TIM-barrel fold that favor effective protein catalysis are discussed.

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Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

Bearne

2020

Chemistry A European J

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Unlike most enzymes, which exhibit stereospecific substrate binding, racemases and epimerases bind and catalyze the reversible interconversion of enantiomeric and epimeric pairs of substrates. Over the past 15 years, a growing number of racemase and epimerase structures have been solved, furnishing insights into the nature of chiral recognition of substrates by these enzymes. Those enzymes catalyzing stereoinversion of a carbon acid substrate through a direct 1,1‐proton transfer mechanism all bind their substrates in a mirror‐image packing orientation. This does not apply generally to racemases and epimerases that use other mechanisms, such as NADH‐dependent epimerases that employ a “flipping” mechanism. In general, polar groups are bound and fixed at the three binding determinants on the protein defining a pseudo‐mirror plane, while nonpolar groups may be mobile. The hydrogen atoms on each stereocenter are positioned antipodal with respect to the pseudo‐mirror plane, making a two‐base mechanism imperative. Recognition that mirror‐image packing is the common binding mode for enantiomeric or epimeric substrates of these enzymes should inform modelling/docking studies and protein engineering.

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Proton Transfer to and from Carbon in Model Reactions

Cited by 9 publications

References 91 publications

Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-¹³C]-Glycolaldehyde in D₂O

Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-¹³C]-Glycolaldehyde in D₂O

Reflections on the catalytic power of a TIM-barrel

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

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Proton Transfer to and from Carbon in Model Reactions

Cited by 9 publications

References 91 publications

Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-13C]-Glycolaldehyde in D2O

Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-13C]-Glycolaldehyde in D2O

Reflections on the catalytic power of a TIM-barrel

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

Contact Info

Product

Resources

About

Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-¹³C]-Glycolaldehyde in D₂O

Hydron Transfer Catalyzed by Triosephosphate Isomerase. Products of the Direct and Phosphite-Activated Isomerization of [1-¹³C]-Glycolaldehyde in D₂O