Linking Conformational Dynamics to Activity Impairment of Disease-associated Human Enzymes

Project Summary/Abstract Motions through a complicated conformational landscape are almost certainly central to enzyme activity. This project aims to test the hypothesis that residue replacements distant from the active site impair activity in human disease-associated missense variants by altering protein dynamics. These proof-of-concept studies will use asparagine synthetase (ASNS) and its variants linked to asparagine synthetase deficiency (ASNSD) as a model system. Leveraging my expertise in mechanistic enzymology, protein dynamics, and the development of high- throughput screening assays, we will combine enzyme kinetics, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and cell-based assays to correlate changes in conformational dynamics with the altered catalytic activities seen for two representative ASNSD-associated variants (ASNSDVs). Time- and temperature- dependent HDX-MS will be used to place the dynamic differences between the wild-type ASNS and the ASNSDVs on a quantitative basis. A cell-based functional complementation assay will also allow us to examine how hydrophobic residue networks permit distal residue changes to affect individual steps in complicated kinetic mechanisms. Project outcomes will clarify how missense mutations that are distal to the enzyme active site lead to activity impairment and will lay a strong foundation for applying this general approach to understand 1,500 human enzymes linked to 2,500 inherited diseases.