On the mechanism of action of methylmalonyl‐CoA mutase

1 When ( methyl‐ 2 H 3 ) methylmalonyl‐CoA was reacted with partially purified methylmalonyl‐CoA mutase, 1 H‐NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion. 2 When [ methyl ‐ 3 H]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′‐ 3 H]adenosylcobalamin to water required the addition of methylmalonyl‐CoA epimerase and of substrate (e.g. succinyl‐CoA). 4 By addition of [ 14 C]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 (2 R )‐ and not (2 S )‐methylmalonyl‐CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using ( 2 H 4 )succinyl‐CoA. 6 When (1‐ 13 C) propionyl‐CoA was reacted with a mixture of highly purified methylmalonyl‐CoA carboxylase, epimerase and mutase, 13 C‐NMR signals were obtained for the thioester carbonyi 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‐ 13 C 2 )Succinic acid and (1,4‐ 13 C 2 )succinyl‐CoA were synthesised and their 13 C‐NMR chemical shifts were exactly determined. 7 Evidence is provided for a strict stereospecificity of the mutase toward the ( 2R )‐epimer of methylmalonyl CoA 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 2 H 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 α‐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.