Human B12-dependent enzymes: Methionine synthase and Methylmalonyl-CoA mutase.

Humans have only two known cobalamin or B12-dependent enzymes: cytoplasmic methionine synthase and mitochondrial methylmalonyl-CoA mutase. A complex intracellular B12 trafficking pathway, comprising a multitude of chaperones, process and deliver cobalamin to the two target enzymes. Methionine synthase catalyzes the transfer of a methyl group from N5-methytetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Cobalamin serves as an intermediate methyl group carrier and cycles between methylcobalamin and cob(I)alamin. Methylmalonyl-CoA mutase uses the 5'-deoxyadenosylcobalamin form of the cofactor and catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. Two chaperones, CblA (or MMAA) and CblB (or MMAB, also known as adenosyltransferase), serve the mutase and ensure that the fidelity of the cofactor loading and unloading processes is maintained. This chapter focuses on assays for purifying and measuring the activities of methionine synthase and methylmalonyl-CoA mutase.