Studies on Methylmalonyl-CoA Mutase from Propionibacterium shermanii

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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“…73), 121 cpm (fr. 7 act nature of this substrate binding has not been elucidated but it is not a subject of this report.…”

Section: H]adenosylcobalaminmentioning

confidence: 99%

“…method of Wood et al [5, 61 as modified in [7]. These were carried out by the coupled spectrophotometric…”

Section: Enzyme Assaysmentioning

confidence: 99%

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

et al. 1986

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“…These were carried out by the coupled spectrophotometric method [2, 31 in a modified form [4], using a Perkin-Elmer 550A UV-Vis spectrometer.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Quantitative measurement of the error in the cryptic stereospecificity of methylmalonyl‐CoA mutase

Michenfelder

Hull

Rétey

1987

European Journal of Biochemistry

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1. Samples of methylmalonyl-CoA and (2H3)methylmalonyl-CoA were prepared by a combination of chemical and enzymic methods. After ion-exchange chromatography the unlabelled methylmalonyl-CoA was pure, the deuterated substance contained 11 -12% dephospho-CoA derivative.2. The sample of unlabelled methylmalonyl-CoA was incubated in deuterated buffer with catalytic amounts of methylmalonyl-CoA mutase, epimerase, and coenzyme B1 2. The progress of the reaction was monitored directly by 'H-NMR spectroscopy at 500 MHz. After equilibrium was established,, a slow mutase-catalysed deuterium incorporation into migratable positions of succinyl-CoA was observed.3. The sample of (2H3)methylmalonyl-CoA was incubated in unlabelled buffer with a mixture of methylmalonyl-CoA mutase, epimerase and coenzyme B12. In withdrawn aliquots, the reaction was interrupted by acidification and the lyophilised samples were examined by 'H-NMR spectroscopy in deuterium oxide. Both rearrangement and protium incorporation into migratable positions of succinyl-CoA were monitored.4. At comparable methylmalonyl-CoA to succinyl-CoA conversion rates, deuterium loss from migratable positions was 4 -6 times faster than the corresponding protium loss. It is confirmed that the stereochemical error of the mutase is amplified by isotope discrimination when deuterium is in migratable positions, whereas it is diminished when protium is in migratable positions.Methylmalonyl-CoA mutase, a coenzyme-B12-dependent enzyme, catalyses the reversible interchange of the CoA thiolester group and a hydrogen atom between two adjacent carbon centers of its substrates, the coenzyme A esters of methylmalonic and succinic acids (Scheme 1). The enzyme is stereospecific for the (2R)-epimer of methylmalonyl-CoA; no reaction was discernible with the (2q-epimer within the limits of experimental error [l]. The (2R)-and (2q-epimers are rapidly interconverted by the coenzyme-B 2-independent methylmalonyl-CoA epimerase via abstraction of the a-proton (OH).

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“…1. This specific activity is comparable to that of Ropionibacterium shermunii [8,9] , which is known as one of the best producers of methylmalonyl-CoA mutase and the extracts of which has the specific activity much higher than in the extract from animal tissues [8,10] . Methylmalonyl-CoA mutase takes an important part in both the conversion of pyruvate to propionate in Propionibacteria and propionate to succinate in animal tissues [ 11 ,121.…”

Section: Conditionsmentioning

confidence: 63%

Methylmalonyl‐CoA mutase in a methanol‐utilizing bacterium, Protaminobacter ruber

1976

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Studies on Methylmalonyl-CoA Mutase from Propionibacterium shermanii

Cited by 43 publications

References 26 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

Quantitative measurement of the error in the cryptic stereospecificity of methylmalonyl‐CoA mutase

Methylmalonyl‐CoA mutase in a methanol‐utilizing bacterium, Protaminobacter ruber

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