Mechanistic Proposals for Vitamin B12 Enzyme Catalysis


The Co-C bond at the centre of all of the Vitamin B12 coenzymes has some very interesting properties, in particular it is a relatively weak bond which can potentially be broken in three ways during enzyme catalysis:

  1. Homolytically to generate a radical and a Co(II)
  2. Heterolytically to form a Co(I) and a carbocation
  3. Heterolytically to form a Co(III) and a carbanion

Mode 3, forming a Co(III) and a carbocation is observed during electrophilic attack on the Co-C bond, for example by Hg(II) or other electrophiles on methylcobalamin.

Thus it is the unique ability of the cobalt atom in the cobalamins (cobalt corrin complexes) to shuttle between three oxidation states, Co(I), Co(II) and Co(III) which is the enabling factor for all of the activities of Vitamin B12. A central issue is just how two different mechaisms have evolved for these various enzymes systems.

To quote Jack Halpern, a pioneer in our understanding of the strength of the Co-C bond: "The accessability of the CoIII, CoII and CoI forms of B12 confers great versatility on CH3 - B12 as a methylating agent, i.e. as a source of CH3-, CH3. and CH3+. Thus CH3 - B12 goes considerably beyond a "Grignard analogue" as a methylating agent."

Homolytic Sission

The reaction whereby a hydrogen and a group on an adjacent carbon atom exchange places 1_2_shift is considered 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 (Electron Magnetic Resonance) 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, 1972}. Abeles et al were the first researchers to propose the radical mechanism. Similar observations have been made for dioldehydrase-substrate, glycerol-dehydrase, ethanol-ammonia lyase, and the ribonucleotide reductase system.

Since it is necessary to rigorously exclude dioxygen from any reaction involving free radicals, a plausible reaction mechanism {described in da Silva and Williams seminal text "The Biological Chemistry of the Elements"} is the following:

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.

The enzymes that bind adenosyl cobablamin, and which catalyses group migrations (mutases) are all believed to be initiated by homolytic cleavage of the Co-C bond, forming an adenosyl radical and with the cobalmin in a Co(II) oxidation state.

There has been a flurry of interest in this subject now that details of the enzyme/coenzyme binding are starting to emerge from mutagenic and X-ray crystal studies of the enzyme.

See Ei-Ichiro Ochiai, for an early discussion of aspects of the radical mechanism. For a more recent discussion of this mechanism, and a description of how cobaloxime models assisted in the understanding, see Lippard and Berg.

Heterolytic Sission

For the methyl transfer reactions involving CH3-B12, it is likely that Co(I) is involved. The reaction catalysed by methionine synthase involves two methyl group transfers:
  1. Me-cob(III)alamin + homocysteine ---> cob(I)alamin + methionine
  2. cob(I)alamin + methyltetrahydrofolate ---> Me-cob(III)alamin + tetrahydrofolate

Recent Molecular Mechanics Interest

Molecular mechanics and molecular dynamics simulations of porphyrins, metalloporphyrins, heme proteins and cobalt corrinoids.

H.M.Marques and K.L. Brown, Coord.Chem. Rev., 2002, 225, 123.

"The attention is focused on the use of molecular mechanics (force field) and molecular dynamics methods for the modeling of the structure of porphyrins (non-planar distortion, metal complexes and ligand interaction, spin states, N-substituent porphyrins, crystal structure effects, "designer" porphyrins), metalloporphyrins and hemoproteins (microperoxidases, Hb, myoglobin, peroxidases, cytochromes), and the cobalt corrinoids (derivs. of vitamin B12)."