Escherichia coli B N5-methyltetrahydrofolate-homocysteine cobalamin methyltransferase: Binding of the folate substrate to the enzyme

Abstract

With the use of 5-methyl[ 14C]tetrahydrofolate and 5-methyltetrahydrofolate-[2- 14C] it was established that the active stereoisomes of the substrate is bound intact to the cobalamin (B 12) methyltransferase. The same amount of binding occurred at 2–4 ° and at 22 °C under conditions in which there was no cobalt methylation, i.e., no reducing system and no S-adenosylmethionine. Also, the recovery of complex preformed at 0 ° and 37 °C was the same upon Sephadex G-25 column filtration; but negligible preformed complex was isolated by column filtration at 22 °C. Under equilibrium conditions the maximum amount of binding observed was 0.81 nmoles of l-5-methyl-[ 14C]tetrahydrofolate/nmole of B 12 enzyme. An equilibrium dissociation constant ( K d ) of 6.2 μ m was calculated and the extrapolated number of binding sites was 0.92/ molecule of bound B 12. The K d is approximately the concentration of l-5-methyltetrahydrofolate which afforded one-half maximal protection to the enzyme against thermal denaturation at 51 °C. It is 5.6-fold less than the K m of 35 μ m for 5-methyl- 14C]tetrahydrofolate in enzymatic transmethylation to homocysteine. Folate has a much lower affinity for the B 12 enzyme than l,5-methyltetrahydrofolate. Although the presence of a B 12 chromophore is not essential for folate substrate binding, the presence or absence of a cobalt-alkyl group significantly effects the amount of complex formed. At all pHs where the B 12 enzyme is soluble, the folate substrate (isoelectric point = 3.89 ± 0.04 SD) binds as an anion with the N 5-aminogroup largely dissociated.