MTHFR is a homotetramer which catalyzes the folate reduction reaction in human bodies using FAD as a cofactor. Mutations occur along the MTHFR catalytic triad disturb the hydride cascade and are hypothesized to cause conformational changes near the substrate-binding site. In this study, Hydrogen-Deuterium Exchange mass spectrometry (HDX-MS) is used to investigate the conformational changes upon substrate binding. Mutations at the 28th position, specifically E28Q and E28D, have been shown by prior kinetic studies to bind to the substrate but not carry out the reduction reaction. Differences in HDX between free and substrate bound enzyme in E28Q and E28D allow us to determine how the substrate binding affects the local conformation. Comparisons of deuterium labels of free and substrate bound enzyme allow the determination of regions of the protein which are impacted by substrate binding. Among the 15 reference peptides identified in this project, residues near the MTHFR protein-protein binding surface as well as the FAD and folate hydrophobic binding pocket show the most changes in HDX, which indicates the substrate binding has an extensive effect on MTHFR conformation. Changes in HDX for both bound E28Q and E28D are consistent with the proposed role of Asp120 and Phe223, which facilitate stabilization of the high-energy intermediate during hydride transfer.

Conformational analysis of substrate bound methylenetetrahydrofolate reductase mutants utilizing hydrogen-deuterium exchange and mass spectrometry

255th National Meeting (2018)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.