Identification of a Rearranged-Substrate, Product Radical Intermediate and the Contribution of a Product Radical Trap in Vitamin B<sub>12</sub> Coenzyme-Dependent Ethanolamine Deaminase Catalysis

Warncke, Kurt; Schmidt, Jennifer C.; Ke, Shyue-Chu

doi:10.1021/ja984005w

Cited by 47 publications

(64 citation statements)

References 55 publications

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“…23 The observation of a product-related radical for ethanolamine in the steady-state suggests that steps subsequent to the formation of the product radical are slower than the 15 N sensitive steps in the catalytic cycle -a scenario consistent with the observation of a small 15 N IE.…”

supporting

confidence: 55%

Probing Nitrogen-Sensitive Steps in the Free-Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase

et al. 2006

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The contribution of C-N bond-breaking/making steps to the rate of the free-radical-mediated deamination of vicinal amino alcohols by adenosylcobalamin-dependent ethanolamine ammonialyase has been investigated by 15 N isotope effects (IE's) and by electron paramagnetic resonance (EPR) spectroscopy. 15 N IE's were determined for three substrates, ethanolamine, (R)-2-aminopropanol, and (S)-2-aminopropanol using isotope ratio mass spectrometry analysis of the product ammonia. Measurements with all three substrates gave measurable, normal 15 N IE's; however, the IE of (S)-2-aminopropanol was ∼ 5-fold greater than the other two. Reaction mixtures frozen during the steady-state show that the 2-aminopropanols give EPR spectra characteristic of the initial substrate radical whereas ethanolamine gives spectra consistent with a product-related radical [Warncke, K.; Schmidt, J. C.; Kee, S.-C., J. Am. Chem. Soc. 1999, 121, 10522-10528]. The steadystate concentration of the radical with (R)-2-aminopropanol is ∼ half that observed with the S isomer, and with (R)-2-aminopropanol the steady-state level of radical is further reduced upon deuteration at C1. The results show that relative heights of kinetic barriers differ among the three substrates such that levels or identities of steady-state intermediates differ. 15 N-Sensitive steps are significant contributors to V/K with (S)-2-aminopropanol.The bacterial enzyme, ethanolamine ammonia-lyase (EAL, EC 4.3.1.7) catalyzes the adenosylcobalamin (AdoCbl)-dependent deamination of ethanolamine or 2-aminopropanols to ammonia and the corresponding aldehyde. 1 The reaction is one of several 1-2 radicalrearrangements/eliminations catalyzed by AdoCbl-dependent enzymes. 2-4 The paradigm for these reactions is an interchange of a group at C2 and a hydrogen atom at C1. With the exception of glutamate mutase, 5 mechanistic details of the migration steps in the AdoCbl-dependent enzymes are not well understood. 3 The EAL reaction can be considered as a C2 → C1 migration of ammonia, followed by decomposition of the product carbinolamine (Scheme 1). 1Migration of ammonia to C1 is not strictly required to form acetaldehyde. 6 The migration mechanism has been demonstrated in the related enzyme dioldehydrase, and derives some support from stereochemistry. 7 Computational studies have indicated that both an internal migration of ammonia or direct elimination are energetically feasible. 6, 8The mechanism includes two H atom abstraction steps that are energetically demanding. Isotopes of H have been used to determine the contributions of these H atom transfers to the overall rate. 1 Transfer of 3 H from 5′ position of AdoCbl to acetaldehyde during turnover shows an isotope effect (IE) of ∼100. ]-ethanolamine is ∼ 6. 9, 13 For (S)-and (R)-2-aminopropanol, D V's are both ∼5. 14 Attenuation of the 2 H IE in the V max suggests that H-insensitive steps in the reaction contribute to limiting of the rate. For example, V/K for the EAL reaction is sensitive to external magnetic fields, suggesting that ra...

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supporting

confidence: 55%

Probing Nitrogen-Sensitive Steps in the Free-Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase

et al. 2006

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“…The kinetically unstable substrate radical intermediate state formed from the aminoethanol substrate can be cryotrapped (Warncke et al, 1999), and is stabilized by storage at 77 K. The forward reaction of the substrate radical ensemble can be synchronously initiated at low temperature by a T -step, and the time-evolution of the reaction is monitored by time-resolved, full-spectrum EPR spectroscopy (Zhu & Warncke, 2008, 2010). The methods have allowed the study of the radical rearrangement and second hydrogen atom transfer (Figure 1, steps 3 and 4) for natural isotopic abundance and 1,1,2,2- 2 H 4 -labeled aminoethanol substrate over the exceptional range of 173–235 K in the bulk frozen solid state (173–187 K range; H. Chen and K. Warncke, unpublished).…”

Section: Low-temperature Frozen Solution Systemmentioning

confidence: 99%

“…The standard procedure for manual cryotrapping of steady-state intermediates in EAL (Warncke et al, 1999; Zhu & Warncke, 2008) starts with manual mixing, on ice, of the prepared EAL-AdoCbl holoenzyme solution (~0.27 ml; in a 12×75 mm disposable culture tube, P/N 14-961-26: Fisher Scientific) with substrate (~0.03 ml of 0.1–1.0 M stock; added with an automatic pipette, under vortex mixing; elapsed time, ~5 s). The sample is then removed from the test tube with a long-stem glass transfer pipette (9 inch Pasteur Pipet, P/N 13-678-6B; Fisher Scientific), and discharged into the middle region of a 4 mm o. d. EPR tube.…”

Section: Low-temperature Frozen Solution Systemmentioning

confidence: 99%

“…Following Co-C bond cleavage, the low-spin Co 2+ (d 7 ; electron spin, S =1/2) in cobalamin interacts with the radical ( S =1/2) to form a radical pair, which is detected by EPR spectroscopy (Gerfen, 1999). The Co 2+ -substrate radical pair state can be formed and cryotrapped in EAL by using 2-aminopropanol (Babior, Moss, Orme-Johnson, & Beinert, 1974) and aminoethanol (Bender, Poyner, & Reed, 2008; Warncke, Schmidt, & Ke, 1999). Figure 2 shows the EPR spectrum of the Co 2+ -aminopropanol substrate radical pair state.…”

Section: Introductionmentioning

confidence: 99%

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Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems

Wang

Zhu

Kohne

et al. 2015

Methods in Enzymology

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Approaches to the resolution and characterization of individual chemical steps in enzyme catalytic sequences, by using temperatures in the cryogenic range of 190–250 K, and kinetics measured by time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy in fluid cryosolvent and frozen solution systems, are described. The preparation and performance of the adenosylcobalamin-dependent ethanolamine ammonia-lyase enzyme from Salmonella typhimurium in the two systems exemplifies the biochemical and spectroscopic methods. General advantages of low-temperature studies are: (1) Slowing of reaction steps, so that measurements can be made by using straightforward T-step kinetic methods and commercial instrumentation, (2) resolution of individual reaction steps, so that first-order kinetic analysis can be applied, and (3) accumulation of intermediates that are not detectable at room temperatures. The broad temperature range from room temperature to 190 K encompasses three regimes: (1) Temperature-independent mean free energy surface (corresponding to native behavior), (2) the narrow temperature region of a glass-like transition in the protein, over which the free energy surface changes, revealing dependence of the native reaction on collective protein/solvent motions, and (3) the temperature range below the glass transition region, for which persistent reaction corresponds to non-native, alternative reaction pathways, in the vicinity of the native configurational envelope. Representative outcomes of low-temperature kinetics studies are portrayed on Eyring and free energy surface (landscape) plots, and guidelines for interpretations are presented.

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“…The spin-spin interaction parameters in EPR spectra of intermediates observed during turnover of S-2-aminopropanol and of ethanolamine by EAL differ from those in spectra of the hydrazine cation radical-cob(II)alamin pair, mainly because of a larger exchange coupling in the former cases [12][13][14]. The distance between Co 2+ and the organic radicals is 10-12 Å ( Figure 6).…”

Section: Weakly Coupled Spin Systemsmentioning

confidence: 95%

The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes

Reed

Mansoorabadi

2003

Current Opinion in Structural Biology

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The radical intermediates generated during the catalytic cycles of adenosylcobalamin-dependent enzymes occur in pairs. The positions of radicals residing on the cofactor, substrate or protein, relative to the position of the low-spin Co 2+ from the cob(II)alamin intermediate, can be extracted from electron paramagnetic resonance (EPR) spectra of the spin-coupled pairs. Examples of radical-Co 2+ pairs that span a range of interspin distances from 3 to 13 Å have been presented. Interspin distances greater than 5 Å require motion of one or more of the participating species. EPR spectroscopy provides a convenient means to determine the structures of these transient intermediates.

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Identification of a Rearranged-Substrate, Product Radical Intermediate and the Contribution of a Product Radical Trap in Vitamin B₁₂ Coenzyme-Dependent Ethanolamine Deaminase Catalysis

Cited by 47 publications

References 55 publications

Probing Nitrogen-Sensitive Steps in the Free-Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase

Probing Nitrogen-Sensitive Steps in the Free-Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase

Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems

The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes

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Identification of a Rearranged-Substrate, Product Radical Intermediate and the Contribution of a Product Radical Trap in Vitamin B12 Coenzyme-Dependent Ethanolamine Deaminase Catalysis

Cited by 47 publications

References 55 publications

Probing Nitrogen-Sensitive Steps in the Free-Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase

Probing Nitrogen-Sensitive Steps in the Free-Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase

Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems

The positions of radical intermediates in the active sites of adenosylcobalamin-dependent enzymes

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Identification of a Rearranged-Substrate, Product Radical Intermediate and the Contribution of a Product Radical Trap in Vitamin B₁₂ Coenzyme-Dependent Ethanolamine Deaminase Catalysis