The Absolute Configuration of Methylmalonyl Coenzyme A and Stereochemistry of the Methylmalonyl Coenzyme A Mutase Reaction

Sprecher, Milon; Clark, M.J.; Sprinson, David B.

doi:10.1016/s0021-9258(18)96846-8

Cited by 71 publications

(5 citation statements)

References 24 publications

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“…It may be noted that cobamide-dependent enzymes do not appear to show any uniformity in regard to the stereochemistry of the reactions they catalyze. Thus in the glutamate mutase (Sprecher et propanediol dehydrase reactions (Zagalak et al, 1966) inversion of substrate configuration occurs, whereas in the methylmalonyl-CoA mutase (Sprecher et al, 1966b) and ribonucleotide reductase of L. leichmannii reaction the configuration of substrate is retained.…”

Section: Discussionmentioning

confidence: 99%

Cobamides and Ribonucleotide Reduction. IV. Stereochemistry of Hydrogen Transfer to the Deoxyribonucleotide^*

Batterham¹,

Ghambeer²,

Blakley³

et al. 1967

Biochemistry

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Section: Discussionmentioning

confidence: 99%

Cobamides and Ribonucleotide Reduction. IV. Stereochemistry of Hydrogen Transfer to the Deoxyribonucleotide^*

Batterham¹,

Ghambeer²,

Blakley³

et al. 1967

Biochemistry

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“…The substrate radical then undergoes intramo- lecular 1,2-transfer of the thioester moiety to form a product radical III. The migrating thioester moiety is subsequently replaced by a hydrogen atom, with retention of configuration (8) to yield product IV. Two plausible alternative mechanisms have been proposed for the 1,2-transfer step, an associative model proceeding via a cyclopropyloxy radical intermediate (9) and a mechanism involving fragmentation to a formyl-CoA radical and acrylate (10).…”

mentioning

confidence: 99%

Protection of Radical Intermediates at the Active Site of Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase^,

2000

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Adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the interconversion of methylmalonyl-CoA and succinyl-CoA via radical intermediates generated by substrate-induced homolysis of the coenzyme carbon-cobalt bond. From the structure of methylmalonyl-CoA mutase it is evident that the deeply buried active site is completely shielded from solvent with only a few polar contacts made between the protein and the substrate. Site-directed mutants of amino acid His244, a residue close to the inferred site of radical chemistry, were engineered to investigate its role in catalysis. Two mutants, His244Ala and His244Gln, were characterized using kinetic and spectroscopic techniques. These results confirmed that His244 is not an essential residue. However, compared with that of the wild type, k(cat) was lowered by 10(2)- and 10(3)-fold for the His244Gln and His244Ala mutants, respectively, while the K(m) for succinyl-CoA was essentially unchanged in both cases. The primary kinetic tritium isotope effect (k(H)/k(T)) for the His244Gln mutant was 1.5 +/- 0.3, and tritium partitioning was now found to be dependent on the substrate used to initiate the reaction, indicating that the rearrangement of the substrate radical to the product radical was extremely slow. The His244Ala mutant underwent inactivation under aerobic conditions at a rate between 1 and 10% of the initial rate of turnover. The crystal structure of the His244Ala mutant, determined at 2.6 A resolution, indicated that the mutant enzyme is unaltered except for a cavity in the active site which is occupied by an ordered water molecule. Molecular oxygen reaching this cavity may lead directly to inactivation. These results indicate that His244 assists directly in the unusual carbon skeleton rearrangement and that alterations in this residue substantially lower the protection of reactive radical intermediates during catalysis.

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“…Methylmalonyl-CoA 1 mutase catalyzes the reversible isomerization of (2R)-methylmalonyl-CoA and succinyl-CoA (1,2). The protein belongs to a group of AdoCbl-dependent enzymes catalyzing unusual 1,2-rearrangements and is the only one found in both bacterial and animal cells (for reviews, see refs 3-5).…”

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confidence: 99%

Stabilization of Radical Intermediates by an Active-Site Tyrosine Residue in Methylmalonyl-CoA Mutase^,

et al. 1998

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The adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the reversible rearrangement of methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of methylmalonyl-CoA mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among methylmalonyl-CoA mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5'-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally between methylmalonyl-CoA and succinyl-CoA, in a ratio of 40:60 when the reaction was initiated by addition of methylmalonyl-CoA and in a ratio of 10:90 when the reaction was initiated by addition of succinyl-CoA. The overall release of tritium was four times faster when succinyl-CoA was used as substrate. The tritium isotope effect on the enzyme catalyzed hydrogen transfer, measured with methylmalonyl-CoA as a substrate, was kH/kT = 30, which is within the expected range for a full primary kinetic tritium isotope effect. The different partitioning of tritium, dependent upon which substrate was used, and the normal value for the kinetic tritium isotope effect contrast markedly with the behavior of wild-type mutase. It appears that the loss of a single interaction involving the hydroxyl group of tyrosine 89 both affects the stability of radical intermediates and decreases the rate of interconversion of the substrate- and product-derived radicals.

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The Absolute Configuration of Methylmalonyl Coenzyme A and Stereochemistry of the Methylmalonyl Coenzyme A Mutase Reaction

Cited by 71 publications

References 24 publications

Cobamides and Ribonucleotide Reduction. IV. Stereochemistry of Hydrogen Transfer to the Deoxyribonucleotide^*

Cobamides and Ribonucleotide Reduction. IV. Stereochemistry of Hydrogen Transfer to the Deoxyribonucleotide^*

Protection of Radical Intermediates at the Active Site of Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase^,

Stabilization of Radical Intermediates by an Active-Site Tyrosine Residue in Methylmalonyl-CoA Mutase^,

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The Absolute Configuration of Methylmalonyl Coenzyme A and Stereochemistry of the Methylmalonyl Coenzyme A Mutase Reaction

Cited by 71 publications

References 24 publications

Cobamides and Ribonucleotide Reduction. IV. Stereochemistry of Hydrogen Transfer to the Deoxyribonucleotide*

Cobamides and Ribonucleotide Reduction. IV. Stereochemistry of Hydrogen Transfer to the Deoxyribonucleotide*

Protection of Radical Intermediates at the Active Site of Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase,

Stabilization of Radical Intermediates by an Active-Site Tyrosine Residue in Methylmalonyl-CoA Mutase,

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Cobamides and Ribonucleotide Reduction. IV. Stereochemistry of Hydrogen Transfer to the Deoxyribonucleotide^*

Cobamides and Ribonucleotide Reduction. IV. Stereochemistry of Hydrogen Transfer to the Deoxyribonucleotide^*

Protection of Radical Intermediates at the Active Site of Adenosylcobalamin-Dependent Methylmalonyl-CoA Mutase^,

Stabilization of Radical Intermediates by an Active-Site Tyrosine Residue in Methylmalonyl-CoA Mutase^,