Substrate binding and activation in cobalamin -independent methionine synthase

Cobalamin-independent methionine synthase (MetE) catalyzes the transfer of the methyl group from methyltetrahydrofolate (CH3-H 4PteGlu3) to the sulfur of homocysteine (Hcy) to make methionine and tetrahydrofolate (H4PteGlu3). This reaction was deemed "impossible" by the chemist Duilio Arigoni, because at physiological pH, the Hcy thiol is not a strong nucleophile and CH3-H4PteGlu 3 provides a very poor leaving group. Hcy has been shown to ligate to a tightly bound zinc in the MetE active site. This interaction activates Hcy by lowering the pKa, such that the thiolate is stabilized at neutral pH.;Perhaps the greater chemical challenge is the activation of CH3 -H4PteGlu3. Protonation of the N5 of CH3 -H4PteGlu3 would produce a good leaving group, but occurs with a pKa of 5 in solution. I hypothesized that protonation should occur upon binding to MetE in the binary or ternary complex or during methyl transfer. I took advantage of the sensitivity of the CH3-H 4PteGlu3 absorbance spectrum to changes in protonation state. Comparison of free and MetE-bound CH3-H4PteGlu 3 absorbance spectra indicated that the N5 is not protonated in the binary complex. Stopped-flow spectrophotometric studies have revealed changes in CH3-H4PteGlu3 absorbance that are consistent with protonation of the substrate. These absorbance changes show saturation with both Hcy and CH3H4PteGlu3, suggesting that the protonation of CH3-H4folate occurs upon formation of the ternary complex and before methyl transfer. These data are consistent with a mechanism in which MetE shifts the pKa values of both substrates to increase the concentration of reactive species at neutral pH. Furthermore, H4PteGlu3 appears to remain bound to MetE, and in the presence of excess Hcy, a MetE•H4PteGlu3•Hcy mixed ternary complex can form, in which H4PteGlu3 appears to be protonated.;The recently determined crystal structure of the MetE•CH3-H 4PteGlu3 binary complex shows the N5-methyl group to be 14 A from the zinc and pointing away from the Zn+2/Hcy site. While the number of evolutionarily conserved interactions between the protein and CH3-H4PteGlu3 suggest that this conformation is biologically relevant, it was not clear whether this binary complex was catalytically competent. Isotope trapping experiments were performed, which demonstrated that 11% and 14% of enzyme-bound CH3-H4PteGlu 3 and Hcy, respectively, could be converted to product, indicating that both binary complexes are chemically competent, but that MetE has a low commitment to catalysis. The equilibrium constants for both substrates binding to form the binary and ternary complexes were also determined, which led to the finding that the substrates bind to MetE synergistically, such that the binding of one substrate increases the affinity for the other.