Evidence that Cobalt−Carbon Bond Homolysis is Coupled to Hydrogen Atom Abstraction from Substrate in Methylmalonyl-CoA Mutase

Padmakumar, R.; Banerjee, Ruma

doi:10.1021/bi962503g

Cited by 156 publications

(204 citation statements)

References 46 publications

Supporting

Mentioning

188

Contrasting

Unclassified

Order By: Relevance

“…[8][9][10][11][12][13][14] C]-adenosylcobalamin was synthesized using established protocols described by Brown et. al, (19) from [8][9][10][11][12][13][14] C]-adenosine that was purchased from Amersham; the radiolabelled material was first converted to 5'-chloroadenosine that was subsequently used to alkylate Cob(I)alamin generated by in situ reduction of hydroxocobalamin with zinc. The resulting [8][9][10][11][12][13][14] C]-AdoCbl was purified by reverse phase HPLC.…”

Section: Experimental Procedures Materialsmentioning

confidence: 99%

Evidence for Coupled Motion and Hydrogen Tunneling of the Reaction Catalyzed by Glutamate Mutase

Cheng

Marsh

2006

Biochemistry

View full text Add to dashboard Cite

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer we previously used presteady state, rapid quench techniques to measure the α-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-3 H]-adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72 respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 ± 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling. Keywordsenzyme; coenzyme-B 12 ; protein-radical; isomerization; transition state; secondary isotope effects Glutamate mutase is one of a group of adenosylcobalamin 1 (AdoCbl, coenzyme B 12 ) dependent enzymes that catalyze unusual carbon skeleton isomerisations. These rearrangements formally involve a 1,2 hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates (1-6). The initial steps of these reactions involve homolysis of the reactive cobalt-carbon bond of the coenzyme to form cob (II)alamin and 5'-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5-deoxyadenosine and substrate radical (or protein radical in the case of AdoCbl-dependent ribonucleotide reductase). These steps have been studied in some detail for several B 12 enzymes and in each case homolysis and hydrogen abstraction are found to be kinetically coupled (7-10), as evidenced by the appearance of a kinetic isotope

show abstract

Section: Experimental Procedures Materialsmentioning

confidence: 99%

Evidence for Coupled Motion and Hydrogen Tunneling of the Reaction Catalyzed by Glutamate Mutase

Cheng

Marsh

2006

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Under steady-state turnover conditions, the intrinsic isotope effect is suppressed, presumably by product release which is believed to be rate limiting. A large deuterium isotope effect has been measured under pre-steady-state conditions when the holoenzyme is rapidly mixed with substrate and formation of the homolysis product, cob(II)alamin, is monitored (Padmakumar et al 1997). Under these conditions, the isotope effect is 35.6 at 20 8C and 49.9 at 5 8C (Chowdhury & Banerjee 2000a,b).…”

Section: Kinetics and Thermodynamics Of The Hydrogen Transfer Stepsmentioning

confidence: 99%

“…Under these conditions, the isotope effect is 35.6 at 20 8C and 49.9 at 5 8C (Chowdhury & Banerjee 2000a,b). These measurements could not be performed with sufficient accuracy at higher temperatures due to the observed rate constant for homolysis being too fast in the presence of unlabelled substrate (Padmakumar et al 1997).…”

Section: Kinetics and Thermodynamics Of The Hydrogen Transfer Stepsmentioning

confidence: 99%

“…Surprisingly, the kinetics of the cobalt-carbon bond homolysis step were found to be sensitive to isotopic substitutions in the substrate (Padmakumar et al 1997) and were interpreted as evidence for kinetic coupling between homolysis and the first hydrogen atom transfer step. A similar sensitivity of the rate of cobalt-carbon bond cleavage to isotopic substitution in the substrate has been seen in other B 12 -dependent enzymes, including glutamate mutase (Marsh & Ballou 1998) and ethanolamine ammonia lyase (Bandarian & Reed 2000).…”

Section: Kinetics and Thermodynamics Of The Hydrogen Transfer Stepsmentioning

confidence: 99%

See 1 more Smart Citation

Quantum catalysis in B ₁₂ -dependent methylmalonyl-CoA mutase: experimental and computational insights

Banerjee

Dybała-Defratyka

Paneth

2006

Phil. Trans. R. Soc. B

Self Cite

View full text Add to dashboard Cite

B 12 -dependent methylmalonyl-CoA mutase catalyses the interchange of a hydrogen atom and the carbonyl-CoA group on adjacent carbons of methylmalonyl-CoA to give the rearranged product, succinyl-CoA. The first step in this reaction involves the transient generation of cofactor radicals by homolytic rupture of the cobalt-carbon bond to generate the deoxyadenosyl radical and cob(II)alamin. This step exhibits a curious sensitivity to isotopic substitution in the substrate, methylmalonyl-CoA, which has been interpreted as evidence for kinetic coupling. The magnitude of the isotopic discrimination is large and a deuterium isotope effect ranging from 35.6 at 20 8C to 49.9 at 5 8C has been recorded. Arrhenius analysis of the temperature dependence of this isotope effect provides evidence for quantum tunnelling in this hydrogen transfer step. The mechanistic complexity of the observed rate constant for cobalt-carbon bond homolysis together with the spectroscopically silent nature of many of the component steps limits the insights that can be derived by experimental approaches alone. Computational studies using a newly developed geometry optimization scheme that allows determination of the transition state in the full quantum mechanical/molecular mechanical coordinate space have yielded novel insights into the strategy deployed for labilizing the cobalt-carbon bond and poising the resulting deoxyadenosyl radical for subsequent hydrogen atom abstraction.

show abstract

“…Co-C5′ bond is kinetically coupled to H atom abstraction from a substrate molecule in the reactions of methylmalonyl-CoA mutase (5), glutamate mutase (6), ethanolamine ammonialyase (7), and diol dehydrase (8). These kinetic experiments have highlighted the transient nature of the 5′-deoxyadenosyl radical species.…”

mentioning

confidence: 98%

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

View full text Add to dashboard Cite

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.

show abstract

Evidence that Cobalt−Carbon Bond Homolysis is Coupled to Hydrogen Atom Abstraction from Substrate in Methylmalonyl-CoA Mutase

Cited by 156 publications

References 46 publications

Evidence for Coupled Motion and Hydrogen Tunneling of the Reaction Catalyzed by Glutamate Mutase

Evidence for Coupled Motion and Hydrogen Tunneling of the Reaction Catalyzed by Glutamate Mutase

Quantum catalysis in B ₁₂ -dependent methylmalonyl-CoA mutase: experimental and computational insights

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

Contact Info

Product

Resources

About

Evidence that Cobalt−Carbon Bond Homolysis is Coupled to Hydrogen Atom Abstraction from Substrate in Methylmalonyl-CoA Mutase

Cited by 156 publications

References 46 publications

Evidence for Coupled Motion and Hydrogen Tunneling of the Reaction Catalyzed by Glutamate Mutase

Evidence for Coupled Motion and Hydrogen Tunneling of the Reaction Catalyzed by Glutamate Mutase

Quantum catalysis in B 12 -dependent methylmalonyl-CoA mutase: experimental and computational insights

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

Contact Info

Product

Resources

About

Quantum catalysis in B ₁₂ -dependent methylmalonyl-CoA mutase: experimental and computational insights