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

Abstract

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.

Metabolism of lactate and sugars by dairy propionibacteria: A review

“…Cobalamin is covalently bound to the enzyme but does not stabilise it [55]. The accepted mode of action of this type of enzyme is the generation of free radicals [105]. The breaking of the Co-C bond of coenzyme B 12 induces the change from its Co3+ form to its C02+ form and a 5' -deoxyadenosyl free radical is generated.…”

Cofactor Editing by the G-protein Metallochaperone Domain Regulates the Radical B12 Enzyme IcmF

“…Acyl-CoA mutases are a group of enzymes that utilize 5Ј-deoxyadenosylcobalamin (AdoCbl 4 or coenzyme B 12 ) as a cofactor for catalyzing carbon skeleton rearrangement reactions (1,2). Isobutyryl-CoA mutase is a member of this enzyme family that is found in bacteria and catalyzes the reversible rearrangement of isobutyryl-and butyryl-CoA mutase (Fig.…”

The error in the cryptic stereospecificity of methylmalonyl‐CoA mutase

“…In particular, signal assignments for a number of reaction products, e. g. non-deuterated succinyl-CHzCoA, were confirmed by homodecouplings carried out between 1.5 h and 2.5 h into the reaction. The assignment and analysis of the complex overlapping multiplet patterns for partially deuterated species were carried out as described previously [3]. We utilised predictions based on the observed approximate additivity of deuterium isotope shifts whereby a geminal deuterium gives an upfield shift of about 0.016 ppm ( z 1.5 3&H) and a vicinal deuterium results in an upfield shift of about 0.006 ppm ( z 0.5 3&H).…”

“…Co. Ltd, Japan) and further purified on a TSK-545 DEAE column (7.5 x 150 mm, LKB Instruments) [3]. Specific activities of 33, 13 and 640 U/mg were obtained for transcarboxylase, mutase and epimerase, respectively.…”

“…The succinyl species are shown in Scheme 2; ('H4)S is tetradeutero-succinyl. The methylmalonyl species I, VI, XIII, ('H4)M contain 0, 1,2, 3 should be generated at the expense of S and 111. Indeed, VIII shows virtually steady-state behaviour over the entire reaction time while S and 111 are consumed.…”