Mechanism of Action of Vitamin B<sub>12</sub>. Ultrafast Radical Clocks Provide No Evidence for Radical Intermediates in Cyclopropane Models for the Methylmalonyl-CoA to Succinyl-CoA Carbon Skeleton Rearrangement

and, Mu He; Dowd, Paul

doi:10.1021/ja971415w

Cited by 28 publications

(17 citation statements)

References 25 publications

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“…In the next step, an interchange of the carbonyl-CoA group and a hydrogen atom occurs between vicinal carbons. The precise mechanism by which the substrate radical isomerizes to the product radical is unresolved, with pathways involving free radical intermediates (3), fragmentation products (11), carbocations (12), carbanions (13), and organocobalt adducts (14,15), each having been proposed (Scheme II). The final step involves reabstraction of a hydrogen atom from deoxyadenosine to generate product and the intact cofactor, AdoCbl.…”

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confidence: 99%

Proton Transfer from Histidine 244 May Facilitate the 1,2 Rearrangement Reaction in Coenzyme B12-dependent Methylmalonyl-CoA Mutase

Maiti

Widjaja

Banerjee

1999

Journal of Biological Chemistry

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Methylmalonyl-CoA mutase is an adenosylcobalamindependent enzyme that catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. This reaction results in the interchange of a carbonyl-CoA group and a hydrogen atom on vicinal carbons. The crystal structure of the enzyme reveals the presence of an aromatic cluster of residues in the active site that includes His-244, Tyr-243, and Tyr-89 in the large subunit. Of these, His-244 is within hydrogen bonding distance to the carbonyl oxygen of the carbonyl-CoA moiety of the substrate. The location of these aromatic residues suggests a possible role for them in catalysis either in radical stabilization and/or by direct participation in one or more steps in the reaction. The mechanism by which the initially formed substrate radical isomerizes to the product radical during the rearrangement of methylmalonyl-CoA to succinyl-CoA is unknown. Ab initio molecular orbital theory calculations predict that partial proton transfer can contribute significantly to the lowering of the barrier for the rearrangement reaction. In this study, we report the kinetic characterization of the H244G mutant, which results in an acute sensitivity of the enzyme to oxygen, indicating the important role of this residue in radical stabilization. Mutation of His-244 leads to an ϳ300-fold lowering in the catalytic efficiency of the enzyme and loss of one of the two titratable pK a values that govern the activity of the wild type enzyme. These data suggest that protonation of His-244 increases the reaction rate in wild type enzyme and provides experimental support for ab initio molecular orbital theory calculations that predict rate enhancement of the rearrangement reaction by the interaction of the migrating group with a general acid. However, the magnitude of the rate enhancement is significantly lower than that predicted by the theoretical studies.

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confidence: 99%

Proton Transfer from Histidine 244 May Facilitate the 1,2 Rearrangement Reaction in Coenzyme B12-dependent Methylmalonyl-CoA Mutase

Maiti

Widjaja

Banerjee

1999

Journal of Biological Chemistry

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“…8). For reactions catalyzed by 2-methyleneglutarate mutase, methylmalonyl-CoA mutase and related acyl-CoA mutases, the inter-conversion of substrate and product radicals most likely occurs through a cyclopropylcarbenyl radical intermediate, a mechanism supported by model chemical reactions [23,24]. In contrast, it has been shown that the rearrangement catalyzed by glutamate mutase involves fragmentation of the glutamyl radical to form a glycyl radical and acrylate followed by recombination to form the methylaspartyl radical [25].…”

Section: Mechanistic Overviewmentioning

confidence: 99%

“…8). Neither species has been directly observed in the enzyme, although studies on model compounds support the latter mechanism [23]. Using the rearrangement of the 3-propanal radical as a minimal model for the methylmalonyl-CoA mutase reaction, the addition-elimination mechanism was found to be the more favorable mechanism by ~12 kcal/mol.…”

Section: Computational Studies Of Adocbl Enzymesmentioning

confidence: 99%

Adenosylcobalamin enzymes: Theory and experiment begin to converge

Marsh

Meléndez

2012

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Adenosylcobalamin (coenzyme B12) serves as the cofactor for a group of enzymes that catalyze unusual rearrangement or elimination reactions. The role of the cofactor as the initiator of reactive free radicals needed for these reactions is well established. Less clear is how these enzymes activate the coenzyme towards homolysis and control the radicals once generated. The availability of high resolution X-ray structures combined with detailed kinetic and spectroscopic analyses have allowed several adenosylcobalamin enzymes to be computationally modeled in some detail. Computer simulations have generally obtained good agreement with experimental data and provided valuable insight into the mechanisms of these unusual reactions. Importantly, atomistic modeling of the enzymes has allowed the role of specific interactions between protein, substrate and coenzyme to be explored, leading to mechanistic predictions that can now be tested experimentally. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

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“…For the reactions catalyzed by methylmalonyl-CoA mutase,[14, 74] isobutyryl-CoA mutase [75] and 2-methyleneglutarate mutase [9, 76] the inter-conversion of substrate and product radicals can occur through a cyclopropylcarbenyl radical intermediate (figure 4), a mechanism supported by model chemical reactions. [77, 78] In contrast, the rearrangement catalyzed by glutamate mutase [18, 19, 79] cannot occur through this type of intermediate and it has been shown experimentally that the mechanism involves fragmentation of the glutamyl radical to form a glycyl radical and acrylate followed recombination to form the methylaspartyl radical. [80]…”

Section: Catalytic Reactions Of Adenosyl Radicalmentioning

confidence: 99%

Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions

2010

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Lead in Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors; ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B12, AdoCbl) or by single-electron reduction of S-adenosylmethionine (AdoMet) complexed to an iron-sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosine’s role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen which rapidly inactivates many radical enzymes. AdoCbl-dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2-hydrogen atom migrations. In contrast, there has been a recent explosion in the number radical AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions. Although all the radical AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes may be active in aerobic organisms including humans.

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Mechanism of Action of Vitamin B₁₂. Ultrafast Radical Clocks Provide No Evidence for Radical Intermediates in Cyclopropane Models for the Methylmalonyl-CoA to Succinyl-CoA Carbon Skeleton Rearrangement

Cited by 28 publications

References 25 publications

Proton Transfer from Histidine 244 May Facilitate the 1,2 Rearrangement Reaction in Coenzyme B12-dependent Methylmalonyl-CoA Mutase

Proton Transfer from Histidine 244 May Facilitate the 1,2 Rearrangement Reaction in Coenzyme B12-dependent Methylmalonyl-CoA Mutase

Adenosylcobalamin enzymes: Theory and experiment begin to converge

Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions

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Mechanism of Action of Vitamin B12. Ultrafast Radical Clocks Provide No Evidence for Radical Intermediates in Cyclopropane Models for the Methylmalonyl-CoA to Succinyl-CoA Carbon Skeleton Rearrangement

Cited by 28 publications

References 25 publications

Proton Transfer from Histidine 244 May Facilitate the 1,2 Rearrangement Reaction in Coenzyme B12-dependent Methylmalonyl-CoA Mutase

Proton Transfer from Histidine 244 May Facilitate the 1,2 Rearrangement Reaction in Coenzyme B12-dependent Methylmalonyl-CoA Mutase

Adenosylcobalamin enzymes: Theory and experiment begin to converge

Adenosyl Radical: Reagent and Catalyst in Enzyme Reactions

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Product

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Mechanism of Action of Vitamin B₁₂. Ultrafast Radical Clocks Provide No Evidence for Radical Intermediates in Cyclopropane Models for the Methylmalonyl-CoA to Succinyl-CoA Carbon Skeleton Rearrangement