Detailed Mechanistic Studies on Covalent Nucleophilic Enzymes Using Time-Resolved Electrospray Mass Spectrometry with H/D Exchange

摘要

1. The combination of TRESI and HDX mass spectrometry is able to show the contrasting conformational dynamics between chymotrypsin (k_(obs) = 0.3916 s~(-1)) and its acyl-enzyme intermediate (k_(obs) = 0.1720 s~(-1)), thus providing evidence that the resting state and catalytically active state have different dynamic behavior. 2. Enzyme dynamics have long been hypothesized to play a role in catalysis. In order to reveal more information about specific dynamics regions of the protein, a site-specific HDX study is necessary. This can be achieved through rapid digestion of the protein, or by nonergodic fragmentation methods like ECD or ETD, directly after labeling with deuterium. 3. Future work will involve online TRESI with HDX followed by rapid proteolysis, for site-specific spatially resolved HDX studies on both OXA-58 and chymotrypsin.