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The reaction whereby a hydrogen and a group on an adjacent carbon atom exchange places 1_2_shift is believed to take place by way of a radical mechanism, such rearrangements are very rare in organic chemistry, indeed the only known instance is the Kharash-Urey reaction. It is especially surprising that the reactions involve specific configuration changes of the substrates. The evidence for a radical mechanism comes principally from observations of EMR spectra for several of the active enzyme systems. Thus if the dioldehydrase (holoenzyme) is treated with the substrate analogue 1,2-propoanediol, a radical signal believed to be due to the 5'-deoxyadenosyl radical, together with a low spin Co(II) signal is formed within milliseconds, and persists whilst there is substrate present {T.H.Finley, J.Valinsky, K.Sato, and R.H.Abeles, J.Biol.Chem., 1972, 247, 4197}. Similar observations have been made for dioldehydrase-substrate, glycerol-dehydrase, ethanol-ammonia lyase, and also the ribonucleotide reductase system. Since it is necessary to rigorously exclude dioxygen from any reaction involving free radicals, a plausible reaction mechanism {proposed in da Silva and Williams seminal text}.

step_1 step_2 Step 1, and the substrate, 1,2-dihydroxyethane, approaches the holoenzyme, and is about to plug the reaction 'bottle'. Then (Step 2) the substrate binds and the cobalt-carbon bond breaks homolytically to give the 5'-adenosyl radical and a Co(II) corrin, anchored to the large coenzyme (are the amide groups involved?) It has been suggested that the cobalt-carbon bond, already a rather weak bond and readily subject to photolysis if the holoenzyme is subjected to visible light, is further weakened by binding of the metalloenzyme to the large coenzyme.

mech_3 mech_4 In Step 3,a hydrogen atom is abstracted from the substrate by the 5'-deoxyadenosyl radical, converting the dangling CH2 into a CH3 Then, Step 4, the enzyme base (B) and the acid (BH+) cause assisted beta-hydroxy fragmentation, that is loss of a water molecule.

mech_5 mech_6 With Step 5, comes the re-addition of a water molecule, resulting in a substrate reorientation of C2 towards the 5'deoxyadenosyl methyl. There must be competing C1 and C2 racemisation. A hydrogen atom abstraction occurs, with competing C2 racemization. The cycle is completed (Step 6), by substrate dehydration and release of ethanal and a water molecule, so that the system can pick up another substrate molecule.

For a more detailed discussion of the mechanism, and a description of how cobaloxime models have assisted in understanding the mechanism, see Lippard and Berg. Ei-Ichiro Ochiai produced an excellent discussion of aspects of the radical mechanism, back in the late 1970's.

For the methyl transfer reactions involving CH3-B12, it is likely that Co(I) is involved, rather than a radical mechanism. More about this later!