Professor Bernard Golding

b.t.golding@ncl.ac.uk

Mechanism of Action of Coenzyme B12 (refs. 1–3)
Coenzyme B12 (adenosylcobalamin) initiates radical chemistry in two types of enzymatic reaction: with the irreversible eliminases (e.g. glycerol dehydratase) and the reversible mutases (e.g. glutamate mutase). In the eliminases the 5'-deoxyadenosyl radical generated by homolysis of the carbon-cobalt-bond of coenzyme B12 moves 11Å away from the cobalt of cob(II)alamin, which acts solely as spectator during the catalytic cycle. In mutases the 5'-deoxyadenosyl radical stays within 3–4Å of the cobalt, with the substrate and product radicals being ca. 3Å further away. It has been suggested that with the mutases, but not eliminases, cob(II)alamin acts as a conductor by stabilising both the 5'-deoxyadenosyl radical and the product-related methylene radical (see figure).

An alternative scenario is that homolysis of the coenzyme's Co—C bond is coupled to substrate activation and so there is no discrete 5'-deoxyadenosyl radical, although cobalt participation is still required. The focus of current research studies at Newcastle is on coenzyme B12-dependent glutamate mutase and 2-methyleneglutarate mutase. Questions to answer are:

  • How is the coenzyme activated and is fission of its Co—C bond coupled to hydrogen atom abstraction from substrate?
  • What are the mechanisms of rearrangements of intermediate radicals and, in particular, what is the precise extent of cobalt participation?

Drug Therapeutics: Novel Anticancer Agents (ref. 4)
The defective functioning of cyclin dependent kinases (CDKs) compromises normal cell cycle progression and is associated with the molecular pathology of cancer. Numerous CDK inhibitors have been reported, the most common being those selective for CDK2 and CDK4. Inhibition of CDK2 may have a therapeutic benefit by stimulating tumour cell apoptosis.

A synthetic strategy developed at Newcastle, employing the Cope elimination (Scheme 1), has enabled the discovery of new, potent inhibitors of CDK2. The nature of inhibitor binding to the CDK2 has been defined by X-ray crystallography (see figure). Ongoing studies are concerned with optimising inhibition of CDK2 and selectively targeting other CDKs.

References

  1. Pierik AJ, Ciceri D, Broker G, Edwards CH, McFarlane W, Winter J, Buckel W, Golding BT, Rotation of the exo-methylene Group of (R)-3-methylitaconate catalyzed by coenzyme B12-dependent 2-methyleneglutarate mutase from Eubacterium barkeri, J. Am. Chem. Soc. 2002, 124, 14039–14048.
  2. Pierik, AJ, Ciceri, D, Lopez, RF, Kroll, F, Bröker, G, Beatrix, B, Bucel, W, Golding BT, Searching for intermediates in the carbon skeleton rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate catalyzed by coenzyme B12-dependent 2-methyleneglutarate mutase from Eubacterium barkeri, Biochemistry, 2005, 44, 10541–10551.
  3. Buckel, W, Kratky, C, Golding BT, Stabilisation of methylene radicals by cob(II)alamin in coenzyme B12-dependent mutases, Chemistry — a European Journal, 2006, 12, 352–362.
  4. Griffin RJ, Henderson A, Curtin NJ, Echalier A, Endicott JA, Hardcastle IR, Newell DR, Noble MEM, Wang LZ, Golding BT, Searching for cyclin-dependent kinase inhibitors using a new variant of the Cope elimination, J. Am. Chem. Soc. 2006, 128, 6012–6013.