Activation of methyltetrahydrofolate by cobalamin-independent methionine synthase

Cobalamin-independent methionine synthase (MetE) catalyzes the final step of de novo methionine synthesis using the triglutamate derivative of methyltetrahydrofolate (CH3-H(4)PteGlu(3)) as methyl donor and homocysteine (Hcy) as methyl acceptor. This reaction is challenging because at physiological pH the Hcy thiol is not a strong nucleophile and CH3-H(4)PteGlu(3) provides a very poor leaving group. Our laboratory has previously established that Hcy is ligated to a tightly bound zinc ion in the MetE active site. This interaction activates Hcy by lowering its pK(a), such that the thiolate is stabilized at neutral pH. The remaining chemical challenge is the activation of CH3-H(4)PteGlu(3). Protonation of N5 of CH3-H-4-PteGlu(3) would produce a better leaving group, but occurs with a pK(a) of 5 in solution. We have taken advantage of the sensitivity of the CH3-H(4)PteGlu(3) absorption spectrum to probe its protonation state when bound to MetE. Comparison of free and MetE-bound CH3-H(4)PteGlu(3) absorbance spectra indicated that the N5 is not protonated in the binary complex. Rapid reaction studies have revealed changes in CH(3)H(4)PteGlu(3) absorbance that are consistent with protonation at N5. These absorbance changes show saturable dependence on both Hcy and CH3-H(4)PteGlu(3), indicating that protonation of CH3-H(4)PteGlu(3) occurs upon formation of the tertiary complex and prior to methyl transfer. Furthermore, the tetrahydrofolate (H-4-PteGlu(3)) product appears to remain bound to MetE, and in the presence of excess Hcy a MetE(.)H(4)PteGlu(3)(.) Hcy mixed ternary complex forms, in which H(4)PteGlu(3) is protonated.