5-methyletrahydrohomofolate: a substrate for cobalamin methyltransferases and an inhibitor of cell growth

5-Methyltetrahydrohomofolate (5-MeH₄-homofolate) is a substrate for the cobalamin (B-12) methyltransferases in Escherichia coli B, rabbit liver, HeLa S₃ cells, and Chinese hamster ovary cells. Each of these B-12 enzymes catalyzes 5-MeH₄-homofolate-homocysteine transmethylation at one-tenth the rate of methionine synthesis from 5-MeH₄-folate. Only one stereoisomer in dl-5-MeH₄-homofolate is active enzymically. Reduced higher 5-alkyl homofolates and folates are weak competitive inhibitors of 5-MeH₄-folate, but are inactive as substrates. The K_m of l-5-MeH₄-homofolate for the E. coli B enzyme (80 μm) is greater than that of 5-MeH₄-folate (35 μm), but its K_m for the Chinese hamster ovary cell transmethylase (20 μm) is less than that of 5-MeH₄-folate (35 μm). l-H₄-Homofolate is a potent competitive inhibitor (K_i = 56 nM) for the E. coli B thymidylate synthetase compared to 5-MeH₄-homofolate (K_i = 56 μM); it is also inactive as a cofactor. However, l-H₄-homofolate is a cofactor for both the HeLa S₃ and Chinese hamster ovary cell thymidylate synthetases, giving 22% of the activity observable with l-H₄-folate. Moreover, its K_m as a cofactor is only 2.1 μm compared to 17 μm for l-H₄-folate. dl-5-MeH₄-Homofolate inhibits the growth of Chinese hamster ovary cells in a reversible manner, but the inhibition is not related to the amount of B-12 holomethyltransferase in the cells.