FURTHER INSIGHTS INTO THE MECHANISM OF ACTION OF METHYLMALONYL-COA MUTASE BY ELECTRON-PARAMAGNETIC-RESONANCE STUDIES

Novel analogues of methylmalonyl-CoA and succinyl-CoA have been prepar ed and used for mechanistic investigations on the coenzyme-B-12-depend ent methylmalonyl-CoA mutase. 1-Carboxyethyl-CoA (1) and 2-carboxyethy l-CoA (2) as well as their sulphoxides (3 and 4) were moderately good inhibitors with K-i values 4-20 times higher than the K-m for succinyl -CoA. 2-Carboxyethyl-CoA 2 and its sulphoxide 4 induced EPR signals wh en bound to the enzyme-coenzyme-B-12 complex. The EPR spectrum of 2 an d its sulphoxide 4 differed very much from those induced by the other substrates. In the case of 2 the EPR spectrum of the holoenzyme/inhibi tor complex showed the presence of an organic radical coupled to cobal (II)amin. The same experiment with 4 leads to the formation of enzyme- bound cobal(II)amin with no detectable organic radical. The analogues 1 and 3 exhibited higher K-i values and did not induce EPR signals bin ding to the enzyme-coenzyme-B-12 complex. Formyl-CoA and acrylate inhi bited 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 substr ate methylmalonyl-CoA. The results are discussed in terms of the mecha nism of the methylmalonyl-CoA mutase reaction.