Chemistry Department Seminar: Elizabeth Trimmer '88, Grinnell College
"Mutational and Conformational Analyses of Folate Binding and Catalysis in E. coli methylenetetrahydrofolate reductase (MTHFR)"
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetra-hydrofolate (CH2-H4folate) to 5-methyltetrahydrofolate (CH3-H4folate) utilizing NAD(P)H as a second substrate and enzyme-bound flavin adenine nucleotide (FAD) as redox coenzyme. The reaction is the sole source of CH3-H4folate, which is used in the synthesis of methionine. In humans, mutations in MTHFR have been correlated with elevated levels of homocysteine, a risk factor for cardiovascular disease, and with neural-tube defects in the fetus. We study MTHFR from Escherichia coli as a model for the catalytic domain of the human enzyme. Because the reaction has a ping pong kinetic mechanism, it can be divided into two half-reactions: a reductive half-reaction where NADH reduces the FAD and an oxidative half-reaction where CH2-H4folate returns the FAD to its oxidized form. In this talk, the focus will be on the oxidative half-reaction, where CH2-H4folate is proposed to accept a proton at the N10 position to open up the imidazolidine ring and form a 5-iminium cation intermediate, which then undergoes reduction by the reduced FAD. The X-ray crystal structure of the Eox•CH3-H4folate complex1 has revealed three active-site amino acid residues - Glu 28, Asp 120, and Phe 223 – with potential roles in folate substrate binding and catalysis in the oxidative half-reaction. To investigate these residues, we have prepared mutant enzymes containing amino acid changes at the site of interest and then measured the kinetic properties of the mutants compared to those of the wild-type enzyme. We have also performed hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the conformational changes upon CH3-H4folate product binding to the wild-type and select mutant enzymes. We hypothesized that Glu28, located next to a structural water molecule near N10 of the folate, could serve as the general acid catalyst to aid 5-iminium cation formation. Consistent with this role, mutant enzymes Glu28Gln and Glu28Asp are able to bind folate, but are completely inactive in catalysis. Differences in HDX between free and CH3-H4folate-bound Glu28Gln, Glu28Asp, and Glu28Gln/Phe223Leu enzymes allow us to determine how folate binding affects the local conformation. Among 13 reference peptides identified, residues near the folate hydrophobic binding pocket as well as near the protein-protein binding surface show the most changes in HDX, consistent with the proposed roles of Asp120 and Phe223 in facilitating folate substrate binding.
*This seminar counts towards the chemistry major seminar attendance requirement.
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