On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution.
When (methyl-2H3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 1H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion. When [methyl-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. 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). By addition of [14C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly. 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 (2H4)succinyl-CoA. When (1-13C) 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 (less than 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 (1,4-13C2)succinyl-CoA were synthesised and their 13C-NMR chemical shifts were exactly determined. 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 2H and 3H 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 alpha-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.
Wölfle
K
K
Michenfelder
M
M
König
A
A
Hull
W E
WE
Rétey
J
J
D000214:Acyl Coenzyme A / Q000378:metabolism; D002384:Catalysis; D003038:Cobamides / Q000378:metabolism; D003903:Deuterium; D007535:Isomerases / Q000378:metabolism*; D007553:Isotope Labeling; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008316:Malonyl Coenzyme A / Q000031:analogs & derivatives / Q000378:metabolism; D008765:Methylmalonyl-CoA Mutase / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D014316:Tritium
D000214:Acyl Coenzyme A; D003038:Cobamides; D014316:Tritium; C015357:methylmalonyl-coenzyme A; D008316:Malonyl Coenzyme A; D003903:Deuterium; C012046:succinyl-coenzyme A; D007535:Isomerases; D008765:Methylmalonyl-CoA Mutase; C000913:cobamamide