Hydrazine Cation Radical in the Active Site of Ethanolamine Ammonia-Lyase:  Mechanism-Based Inactivation by Hydroxyethylhydrazine

Bandarian, Vahe; Reed, George H.

doi:10.1021/bi990620g

Cited by 51 publications

(89 citation statements)

References 33 publications

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“…Overstabilization of this intermediate may prevent it from reacting further and reabstracting a hydrogen from deoxyadenosine, to regenerate Ado ⅐ and eventually the resting state of the coenzyme. This type of mechanism-based inactivation has previously been observed with ethanolamine ammonia lyase following incubation with (i) hydroxyethylhydrazine (leading to formation of stable hydrazine radical) (29) and (ii) glycolaldehyde (leading to formation of a cis-ethanesemidione anion radical) (30). The stable radical is likely to be one of the three PLP-bound forms shown in Scheme 2.…”

Section: Cob(ii)alamin Is Formed Only Transiently Upon Reactionmentioning

confidence: 55%

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

Wolthers

Rigby

Scrutton

2008

Journal of Biological Chemistry

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We report an analysis of the reaction mechanism of ornithine 4,5-aminomutase, an adenosylcobalamin ( ; coenzyme B 12 ) serves as a radical repository for a group of enzymes that catalyze unusual isomerizations, whereby a hydrogen atom (H) is interchanged with an electron-withdrawing group (X) on a neighboring carbon atom (Scheme 1) (1-4). To date, 11 AdoCbl-dependent isomerases have been identified and grouped into three classes: (i) Class I (mutases) that catalyze carbon skeletal rearrangements; (ii) Class II (eliminases) that catalyze, with one exception, the migration and elimination of a heteroatom; and (iii) Class III (aminomutases) that catalyze intramolecular 1,2-amino shifts. Turnover for all AdoCbl-dependent isomerases begins with substrate-induced homolysis of the AdoCbl Co-C bond and formation of two paramagnetic centers: the 5Ј-deoxyadenosyl radical (Ado ⅐ ) and cob(II)alamin. The highly reactive Ado ⅐ species propagates radical formation by abstracting a hydrogen atom from the substrate (or an amino acid side chain in the case of ribonucleotide reductase) (5), generating deoxyadenosine and a substrate radical. The latter carbon-centered radical isomerizes to a product radical intermediate, which then reabstracts a hydrogen atom to form Ado ⅐ . Geminate recombination between Ado ⅐ and cob(II)alamin regenerates AdoCbl and primes the enzyme for another catalytic cycle.Studies on AdoCbl-dependent isomerases have shown that the first step in the catalytic cycle (homolytic rupture of the Co-C bond) is coupled kinetically to hydrogen abstraction by Ado ⅐ (6 -9). Thus, the highly reactive Ado ⅐ species is short lived due to rapid neutralization by hydrogen abstraction, and this species has yet to be observed. The second paramagnetic center, cob(II)alamin, "lingers" in the active site, until product is formed, after which it recombines with Ado ⅐ to form the resting enzyme. In the presence of substrate, cob(II)alamin accumulates at a steady-state concentration, which can be observed by EPR and UV-visible spectroscopy. Class I and Class II isomerases have been extensively studied, and the cob(II)alamin spectroscopic signature has been valuable in studies of mechanism (6,8,10). Lysine 5,6-aminomutase (5,6-LAM) is the only Class III enzyme for which detailed studies of mechanism are reported. Cob(II)alamin does not accumulate in steady-state turnover as the enzyme undergoes rapid "suicide inactivation" involving removal of an electron from cob(II)alamin by a substrate and/or product radical intermediate (11). Irreversible formation of cob(III)alamin results, and cob(II)alamin is unable to recombine with Ado ⅐ to reform AdoCbl in the resting form of the enzyme.

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Section: Cob(ii)alamin Is Formed Only Transiently Upon Reactionmentioning

confidence: 55%

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

Wolthers

Rigby

Scrutton

2008

Journal of Biological Chemistry

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“…Euler rotations (29) were therefore used to express each tensor in a common reference frame, in this case the g-axis system of Co 2+ . The strategy used to diagonalize the energy matrix and simulate the field-swept powder EPR spectrum was described previously (30). In this analysis, the 59 Co-hyperfine interaction is treated to first-order, effectively rendering the energy matrix block-diagonal in m I .…”

Section: Spectral Simulationsmentioning

confidence: 99%

Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate

et al. 2006

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We have investigated the reaction of glutamate mutase with the glutamate analog, 2-thiolglutarate. In the standard assay, 2-thiolglutarate behaves as a competitive inhibitor with K i = 0.05 mM. However, rather than simply binding inertly at the active site, 2-thiolglutarate elicits cobalt-carbon bond homolysis and the formation of 5′-deoxyadenosine. The enzyme exhibits a complicated EPR spectrum in the presence of 2-thiolglutarate that is markedly different from any previously observed with the enzyme. The spectrum was well simulated by assuming that it arises from electron-electron spin coupling between a thioglycolyl radical and low-spin Co 2+ in cob(II)alamin. Analysis of the zero-field splitting parameters obtained from the simulations places the organic radical at ∼ 10 Å from the cobalt, and at a tilt angle of ∼ 70° to the normal of the corrin ring. This orientation is in good agreement with that expected from the crystal structure of glutamate mutase complexed with substrate. 2-thiolglutarate appears to react in a manner analogous to glutamate by first forming a thioglutaryl radical at C-4 that then undergoes fragmentation to produce acrylate and the sulfurstablized thioglycolyl radical. The thioglycolyl radical accumulates on the enzyme suggesting it is too stable to undergo further steps in the mechanism at a detectable rate. Keywordsenzyme; coenzyme-B 12 ; free radicals; isomerization; substrate analog; EPR spectrometry Adenosylcobalamin 1 (Coenzyme B 12 , AdoCbl) is the coenzyme for a group of enzymes that catalyze unusual rearrangement or elimination reactions, as well as for class II ribonucleotide reductases. In these enzymes, the coenzyme serves as a masked form of the 5′-deoxyadenosyl radical that is generated through homolytic fission of the AdoCbl cobalt-carbon bond (1-6). An interesting and still poorly understood aspect of these reactions is how these enzymes catalyze homolysis of the coenzyme because the generation of free radicals from stable, closed shell molecules is energetically highly unfavorable.In all cases, homolysis of AdoCbl is tightly coupled to the formation of substrate-based radicals (or in the case of ribonucleotide reductase, a protein-based thiyl radical). The initially formed 5′-deoxyadenosyl radical is extremely unstable and has never been observed spectroscopically.

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“…Energy levels and transition energies were obtained by diagonalization of the energy matrix. Field-swept powder EPR spectra were calculated as described previously (34). In this analysis, the 59 Co-hyperfine interaction was treated to first-order, effectively reducing the energy matrix to a block diagonal form in the 59 Co nuclear quantum number, m I .…”

Section: Spectral Simulationsmentioning

confidence: 99%

Analysis of the Cob(II)alamin−5‘-Deoxy-3‘,4‘-anhydroadenosyl Radical Triplet Spin System in the Active Site of Diol Dehydrase

et al. 2006

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A triplet spin system (S = 1) is detected by low-temperature electron paramagnetic resonance (EPR) spectroscopy in samples of diol dehydrase and the functional adenosylcobalamin (AdoCbl) analog 5′-deoxy-3′,4′-anhydroadenosylcobalamin (anAdoCbl). Different spectra are observed in the presence and absence of the substrate (R,S)-1,2-propanediol. In both cases, the spectra include a prominent half-field transition (ΔM S = 2) that is a hallmark of strongly-coupled triplet spin systems. The appearance of 59 Co-hyperfine splitting in the EPR signals as well as the positions (g-values) of the signals in the spectra show that half of the triplet spin is contributed by the low spin Co 2+ of cob (II)alamin. Linewidth effects from isotopic labeling ( 13 C and 2 H) in the 5′-deoxy-3′,4′-anhydroribosyl ring demonstrate that the other half of the spin triplet is from an allylic 5′-deoxy-3′, 4′-anhydroadenosyl (anhydroadenosyl) radical. The zero-field splitting (ZFS) tensors describing the magnetic dipole-dipole interactions of the component spins of the triplets have rhombic symmetry because of electron spin delocalization within the organic radical component and nearness of the radical to the low spin Co 2+ . The dipole-dipole interaction was modeled as a summation of pointdipole interactions involving the spin-bearing orbitals of the anhydroadenosyl radical and cob(II) alamin. Geometries which are consistent with the ZFS tensors in the presence and absence of substrate position the 5′-carbon of the anhydroadenosyl radical 3.5 Å and 4.1 Å and from Co 2+ , respectively. Homolytic cleavage of the cobalt-carbon bond of the analog in the absence of substrate indicates that, in diol dehydrase, binding of the coenzyme to the protein weakens the bond prior to binding of substrate.A common characteristic of AdoCbl 1 -dependent enzymes is the generation and utilization of enzyme-bound organic radicals (1-3). The Co-C5′ bond of AdoCbl is relatively weak having a bond dissociation energy of ~ 30 kcal mol −1 (4). A prevalent mechanism for AdoCbldependent enzymes begins with the homolytic cleavage of the Co-C5′ bond to give cob(II) alamin and the 5′-deoxyadenosyl radical (1). The 5′-deoxyadenosyl radical being an unstabilized, primary alkyl radical is expected to have a short lifetime, low concentration, and a high reactivity towards H atom abstraction. Thus far, these properties of the 5′-deoxyadenosyl radical have precluded direct observation of the radical by EPR spectroscopy.

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Hydrazine Cation Radical in the Active Site of Ethanolamine Ammonia-Lyase: Mechanism-Based Inactivation by Hydroxyethylhydrazine

Cited by 51 publications

References 33 publications

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

Mechanism of Radical-based Catalysis in the Reaction Catalyzed by Adenosylcobalamin-dependent Ornithine 4,5-Aminomutase

Reaction of Adenosylcobalamin-Dependent Glutamate Mutase with 2-Thiolglutarate

Analysis of the Cob(II)alamin−5‘-Deoxy-3‘,4‘-anhydroadenosyl Radical Triplet Spin System in the Active Site of Diol Dehydrase

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