Investigation of the Mechanism of the Methylmalonyl-CoA Mutase Reaction with the Substrate Analogue: Ethylmalonyl-CoA

Rétey, János; Smith, Edward H.; Zagalak, B.

doi:10.1111/j.1432-1033.1978.tb12110.x

Cited by 37 publications

(21 citation statements)

References 48 publications

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“…Parallel experiments using the substrate analogue ethylmalonyl-CoA partially confirmed the above results [3]. Here mass spectrometry showed however exclusively monodeuterated methylsuccinate species produced by the combined action of mutase/epimerase in deuterium oxide [3].…”

Section: Kinetic Experiments With Unlabelled ( = H 4 ) and ( 2 R3 R)supporting

confidence: 68%

“…J., unpublished results cited by Arigoni and Eliel [2]) and ethylmalonyl-CoA 131 as substrates. An examination with the homologous substrate showed, furthermore, that the substitution at the second centre occurs also mainly with retention [3]. The incomplete stereospecificity of this reaction was accompanied by partial transfer of a heavy hydrogen isotope (2H or 3H) from a migratable position to the medium while no measurable incorporation in the opposite direction occurred when the medium was labelled.…”

mentioning

confidence: 96%

“…Here mass spectrometry showed however exclusively monodeuterated methylsuccinate species produced by the combined action of mutase/epimerase in deuterium oxide [3]. A 'H-NMR spectrum at higher resolution and sensitivity than that first reported [3] revealed that the distribution of the deuterium between the diastereotopic positions of the methylene group (HS,R/HReR, for stereochemical nomenclature see [29]), was 94:6 (Konig, A. and Retey, J., unpublished) [30].…”

Section: Kinetic Experiments With Unlabelled ( = H 4 ) and ( 2 R3 R)mentioning

confidence: 99%

“…The use of stereospecifically as well as fully deuterated ethylmalonyl-CoA species as mutase substrates showed for the first time that a considerable portion (~4 0 % ) of the migrating deuterium was lost [3]. With the corresponding tritiated derivative as much as 85% of the label in migratable positions was washed out into water [30, 311. Results of the present work extend these findings also to the natural substrate methylmalonyl-CoA and provide evidence that both mutase and epimerase are required for the washing-out process.…”

Section: Kinetic Experiments With Unlabelled ( = H 4 ) and ( 2 R3 R)mentioning

confidence: 99%

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On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

et al. 1986

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1. When (methyL2H 3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 'H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion.2. When [methyf-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. 3. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA).4. By addition of ['4C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly.5. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)-and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using ('H4)succinyl-CoA.6 . When (I -I3C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (< 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place.(1 ,4-13C2)Succinic acid and (1 ,4-'3C2)succinyl-CoA were synthesised and their I3C-NMR chemical shifts were exactly determined. 7. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonylCoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3 H to water and slow washing-in from the medium. Whenever migration of protium from the sterically less preferred 3-pro(S)-position of succinyl-CoA occurs and simultaneously a heavy isotope is maneuvered from the migratable 3-pro(R)-position into the labile a-position of methylmalonyl-CoA, the substitution by the COSCoA group takes place with inversion of configuration. When the sterically preferred 3-pro(R)-hydrogen atom migrates, the previously reported stereochemical retention occurs.A mechanistic and stereochemical scheme is discussed that fully accounts for all observations.

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Section: Kinetic Experiments With Unlabelled ( = H 4 ) and ( 2 R3 R)supporting

confidence: 68%

mentioning

confidence: 96%

Section: Kinetic Experiments With Unlabelled ( = H 4 ) and ( 2 R3 R)mentioning

confidence: 99%

Section: Kinetic Experiments With Unlabelled ( = H 4 ) and ( 2 R3 R)mentioning

confidence: 99%

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On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

et al. 1986

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“…We tested two analogs with extended carbon skeletons: ethylmalonyl-CoA, a poor alternative substrate for wild-type enzyme (20,21), and allylmalonyl-CoA, which is predicted to stabilize the substrate allylic radical. With wild-type enzyme, both analogs act as competitive inhibitors and exhibit similar K i values (4 50 μM) ( Table I).…”

Section: Inactivation Of Y243a By Substrate Analogsmentioning

confidence: 99%

Alternative Pathways for Radical Dissipation in an Active Site Mutant of B₁₂-Dependent Methylmalonyl-CoA Mutase

Padovani

Banerjee

2006

Biochemistry

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Methylmalonyl-CoA mutase catalyzes the adenosylcobalamin-dependent rearrangement of (2R)-methylmalonyl-CoA to succinyl-CoA. The crystal structure of the enzyme reveals that Y243 is in van der Waals contact with the methyl group of the substrate and suggests a possible role for it in the stereochemical control of the reaction. This hypothesis was tested by designing a molecular hole by replacing the phenolic side chain of Y243 with the methyl group of alanine. The Y243A mutation lowered the catalytic efficiency >4 × 10 4 -fold compared to wild type enzyme, the K Mapp for the cofactor ~4-fold, and the cob(II)alamin concentration under steady-state turnover conditions ~2-fold. However, the mutation did not appear to lead to loss of the stereochemical preference for the substrate. The Y243A mutation is expected to create a cavity and should in principle, allow accommodation of bulkier substrates. To test this, we used ethylmalonyl-CoA and allylmalonyl-CoA, as alternate substrates. Surprisingly, both analogs resulted in suicidal inactivation albeit in an O 2 -dependent and O 2 -independent fashion, respectively. The inactivation by allylmalonyl-CoA was further investigated and revealed formation of cob(II)alamin at a ~1.5-fold higher rate than with wild-type mutase under single turnover conditions. Product analysis revealed a stoichiometric mixture of 5′-deoxyadenosine, aquocobalamin and allylmalonyl-CoA. Taken together, these results are consistent with an internal electron transfer from cob(II)alamin to the substrate analog radical. These studies serve to emphasize the fine control exerted by Y243 in the vicinity of the substrate to minimize radical extinction in side reactions.Methylmalonyl-CoA mutase catalyzes the reversible isomerization of methylmalonyl-CoA to succinyl-CoA and is dependent on 5′-deoxyadenosylcobalamin (AdoCbl) 1 or coenzyme B 12 for activity (Figure 1) (1). In this reaction, the AdoCbl cofactor is used as a radical reservoir that generates via homolysis of theCo-carbon bond, a pair of radicals: cob(II)alamin and a reactive 5′-deoxyadenosyl radical (Ado•). The latter abstracts a hydrogen atom from the substrate generating a substrate-centered radical that, in turn, rearranges to a product-centered radical. The remainder of the catalytic cycle is completed by reversal of the sequence of steps in the first half of the reaction (Figure 1).Two questions of long-standing interest regarding AdoCbl-dependent enzymes are how the inherent kinetic inertness of the Co-carbon bond is overcome to effect a trillion-fold acceleration in the homolysis rate (2) and how the high reactivity of radical intermediates are controlled to minimize side reactions.

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MethylmalonylCoAMutase

Gruber

Kratky

2004

Handbook of Metalloproteins

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The 5′‐adenosylcobalamin dependent methylmalonyl‐CoA mutase (MCM) catalyzes the reversible and stereospecific equilibration of ( 2R )‐methylmalonyl CoA and succinyl CoA through a radical mechanism. Its occurrence ranges from bacteria to mammals, where it is required for the metabolism or biosynthesis of propionate. MCM from Propionibacterium shermanii is an αβ‐heterodimer of total molecular weight 150 kDa, containing an inactive β‐subunit along with an active α‐subunit, the latter binding one adenosylcobalamin molecule close to the active site. The protein was found to occur in two fundamentally different conformations: in the presence of substrates it crystallizes in a “closed” conformation, whereas in the absence of substrates an “open” conformation is observed. This dramatic conformational change suggests a mechanism for the first step of the MCM‐catalyzed reaction cycle, i.e. the substrate‐induced homolysis of the Co–C bond.

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Investigation of the Mechanism of the Methylmalonyl-CoA Mutase Reaction with the Substrate Analogue: Ethylmalonyl-CoA

Cited by 37 publications

References 48 publications

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

Alternative Pathways for Radical Dissipation in an Active Site Mutant of B₁₂-Dependent Methylmalonyl-CoA Mutase

MethylmalonylCoAMutase

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Investigation of the Mechanism of the Methylmalonyl-CoA Mutase Reaction with the Substrate Analogue: Ethylmalonyl-CoA

Cited by 37 publications

References 48 publications

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

Alternative Pathways for Radical Dissipation in an Active Site Mutant of B12-Dependent Methylmalonyl-CoA Mutase

MethylmalonylCoAMutase

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Alternative Pathways for Radical Dissipation in an Active Site Mutant of B₁₂-Dependent Methylmalonyl-CoA Mutase