Novel analogues of methylmalonyl-CoA and succinyl-CoA have been prepared and used for mechanistic investigations on the coenzyme-B ,,-dependent methylmalonyl-CoA mutase. 1 -Carboxyethyl-CoA(1) and 2-carboxyethyl-CoA (2) as well as their sulphoxides (3 and 4) were moderately good inhibitors with K, values 4-20 times higher than the K, for succinyl-CoA. 2-Carboxyethyl-CoA (2) and its sulphoxide 4 induced EPR signals when bound to the enzyme-coenzyme-B,, complex. The EPR spectrum of -2 and its sulphoxide 4 differed very much from those induced by the other substrates. In the case of 2 the EPR spectrum of the holoenzymelinhibitor complex showed the presence of an organic radical coupled to cobal(I1)amin. The same experiment with 9 leads to the formation of enzyme-bound cobal(I1)amin with no detectable organic radical. The analogues 1 and 3 exhibited higher K, values and did not induce EPR signals binding to the enzyme-coenzyme-B,, complex.Formyl-CoA and acrylate inhibited the enzyme synergistically but were unable to induce EPR signals and to form the product. Ethylmalonyl-CoA, known as a poor substrate, induced a similar but less intense EPR signal than the natural substrate methylmalonyl-CoA. The results are discussed in terms of the mechanism of the methylmalonyl-CoA mutase reaction.Keywords: coenzyme B ; methylmalonyl-CoA analogs ; EPR spectra; inhibition kinetics.The coenzyme-B,,-dependent methylmalonyl-CoA mutase catalyses the rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA (Eqn 1) [I, 21. As for other coenzyme-B,,-dependent enzymes catalysing carbon skeleton rearrangements, e.g. 2-methyleneglutarate mutase [3] and glutainate mutase [4], the catalytic reaction is initiated by homolysis of the Co-C bond of the coenzyme which occurs upon binding the substrate to the holoenzyme and results in the formation of a highly reactive 5'-deoxyadenosyl radical and the paramagnetic Coz+. The 5'-deoxyadenosyl radical abstracts a hydrogen atom from the (nonactivated) methyl group of methylmalonyl-CoA and the thus generated substrate radical rearranges to the product radical which abstracts the hydrogen atom back from the transiently formed 5'-deoxyadenosine. Thus, during the rearrangement, several organic radical species and the paramagnetic cob(I1)alamin are postulated as intermediates (Scheme 1). Recent EPR investigations using methylmalonyl-CoA [ 5 ] , its dethia(carba) derivatives and the inhibitor 3-carboxypropyl-CoA (61 as a substrate documented the formation of Co2+ in the frozen steady-state of the enzymic reaction. The characterisation of organic radicals however failed because the EPR signals were too complex. The X-ray structure of the coenzyme-B ,,-binding fragment of the methionine synthetase (71 revealed the replacement of the dimethylbenzimidazole of the coenzyme by a His residue of the protein. The co-ordination of a His to the Co2+ was then confirmed for glutamate mutase [ 81 and methylmalonyl-CoA mutase [9] by incorporating "N-labelled histidine into the enzymes followed by EPR measurements. Recently...

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