Mechanism of action of coenzyme B12. Hydrogen transfer in the isomerization of .beta.-methylaspartate to glutamate

Eagar, Robert G.; Baltimore, Barbara G.; Herbst, M.; Barker, H. A.; Richards, John H.

doi:10.1021/bi00752a017

Cited by 34 publications

(17 citation statements)

References 38 publications

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“…Substrates. L-threo-3-Methylaspartate and L-threo-[3-2 H 3methyl]aspartate were prepared enantiomerically pure by enzymic synthesis (16). D,L-Glutamic acid was purchased from Sigma Chemical Company and D,L-[2,4,4-2 H 3 ]glutamic acid from Cambridge Isotope Laboratories Inc. Because deuterated glutamate was available only in racemic form, we used racemic protiated glutamate in these experiments for comparative purposes.…”

Section: Methodsmentioning

confidence: 99%

Coupling of Cobalt−Carbon Bond Homolysis and Hydrogen Atom Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase

1998

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Adenosylcobalamin-dependent glutamate mutase catalyzes an unusual carbon skeleton rearrangement that proceeds through the formation of free radical intermediates generated by the substrate-induced cleavage of the coenzyme cobalt-carbon bond. The reaction was studied at 10 degrees C with various concentrations of L-glutamate and L-threo-3-methylaspartate and with use of stopped-flow spectroscopy to follow the formation of cob(II)alamin. Either substrate induces rapid formation of cob(II)alamin, which accumulates to account for about 25% of the total enzyme species in the steady state when substrate is saturating. Measurements of the rate constant for the formation of cob(II)alamin demonstrate that the enzyme accelerates the rate of homolysis of the cobalt-carbon bond by at least 10(12)-fold. Very large isotope effects on cob(II)alamin formation, of 28 and 35, are observed with deuterated L-glutamate and deuterated L-threo-3-methylaspartate, respectively. This implies a mechanism in which Co-C bond homolysis is kinetically coupled to substrate hydrogen abstraction. Therefore, adenosyl radical can only be formed as a high-energy intermediate only at very low concentrations on the enzyme. The magnitude of the isotope effects suggests that hydrogen tunneling may play an important role catalysis.

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Section: Methodsmentioning

confidence: 99%

Coupling of Cobalt−Carbon Bond Homolysis and Hydrogen Atom Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase

1998

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“…[70] The barrier for this conversion was estimated as about 63 kJ mol À1 from the large isotope effect observed for the hydrogen transfer from 5'-deoxyadenosine to the product methylene radical in ethanolamine ammonia lyase. [79,80] [67,81] whereas hydrogen abstraction from an isolated methyl group should be more difficult than from a methylene group adjacent to a carboxylate moiety. This observation is consistent with the proposed stabilisation of the 3-methyleneaspartate radical by cob(ii)alamin and may result in an activation energy significantly less than 63 kJ mol was mainly attributed to a large secondary equilibrium isotope effect resulting from the labelling of the 5'-carbon atom of deoxyadenosine with deuterium during turnover.…”

Section: Reversible Formation and Stabilisation Of Methylene Radicalsmentioning

confidence: 99%

Stabilisation of Methylene Radicals by Cob(II)alamin in Coenzyme B₁₂ Dependent Mutases

Buckel

Kratky

Golding

2005

Chemistry A European J

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Coenzyme B12 initiates radical chemistry in two types of enzymatic reactions, the irreversible eliminases (e.g., diol dehydratases) and the reversible mutases (e.g., methylmalonyl-CoA mutase). Whereas eliminases that use radical generators other than coenzyme B12 are known, no alternative coenzyme B12 independent mutases have been detected for substrates in which a methyl group is reversibly converted to a methylene radical. We predict that such mutases do not exist. However, coenzyme B12 independent pathways have been detected that circumvent the need for glutamate, beta-lysine or methylmalonyl-CoA mutases by proceeding via different intermediates. In humans the methylcitrate cycle, which is ostensibly an alternative to the coenzyme B12 dependent methylmalonyl-CoA pathway for propionate oxidation, is not used because it would interfere with the Krebs cycle and thereby compromise the high-energy requirement of the nervous system. In the diol dehydratases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt bond of coenzyme B12 moves about 10 A away from the cobalt atom in cob(II)alamin. The substrate and product radicals are generated at a similar distance from cob(II)alamin, which acts solely as spectator of the catalysis. In glutamate and methylmalonyl-CoA mutases the 5'-deoxyadenosyl radical remains within 3-4 A of the cobalt atom, with the substrate and product radicals approximately 3 A further away. It is suggested that cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product-related methylene radicals.

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“…In anaerobes the pathways of substrate conversion are often quite different from those found in aerobic organisms. Examples are the anaerobic catabolism of glutamate (43,88,154,612,633,634,688,689), glycine (34,33,100,(311)(312)(313)(314), lysine (35,72,108,138,276,531,623,659,735), ornithine (277), and purines (42,554,578). The difference of pathways is necessitated by the thermodynamic requirements imposed on anaerobic metabolism and by the need for compensation of the hydrogen balance via intermediate product coupling as discussed above.…”

Section: Vol 41 1977mentioning

confidence: 99%

Energy conservation in chemotrophic anaerobic bacteria.

Thauer¹,

Jungermann²,

Decker³

1977

Bacteriological Reviews

2,460

1,166

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Mechanism of action of coenzyme B12. Hydrogen transfer in the isomerization of .beta.-methylaspartate to glutamate

Cited by 34 publications

References 38 publications

Coupling of Cobalt−Carbon Bond Homolysis and Hydrogen Atom Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase

Coupling of Cobalt−Carbon Bond Homolysis and Hydrogen Atom Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase

Stabilisation of Methylene Radicals by Cob(II)alamin in Coenzyme B₁₂ Dependent Mutases

Energy conservation in chemotrophic anaerobic bacteria.

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Mechanism of action of coenzyme B12. Hydrogen transfer in the isomerization of .beta.-methylaspartate to glutamate

Cited by 34 publications

References 38 publications

Coupling of Cobalt−Carbon Bond Homolysis and Hydrogen Atom Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase

Coupling of Cobalt−Carbon Bond Homolysis and Hydrogen Atom Abstraction in Adenosylcobalamin-Dependent Glutamate Mutase

Stabilisation of Methylene Radicals by Cob(II)alamin in Coenzyme B12 Dependent Mutases

Energy conservation in chemotrophic anaerobic bacteria.

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Stabilisation of Methylene Radicals by Cob(II)alamin in Coenzyme B₁₂ Dependent Mutases