| Description |
In mitochondria, the tricarboxylic acid (TCA) cycle integrates oxidation pathways of all major carbon sources by eight enzymes that are associated into a supramolecular complex called the metabolon. In metabolons, substrate channeling is the most remarkable feature, which is the directed or facilitated intermediate transport between active sites. Considerable studies have been done on biological implications of substrate channeling in improving coupled catalysis. Nowadays the TCA cycle metabolon attracts increasing interest from experts in the field of bioelectrocatalysis, who have been seeking highly efficient enzyme cascades enabling deep substrate oxidation and promoted mass transport. This dissertation research focuses on the fundamental understanding of the TCA cycle metabolon from mechanistic and structural perspectives. In this work, a microfluidic system was built to study enzyme dynamics in metabolon formation and showed that intermediate generated by one enzyme induces directed transport of subsequent enzymes against the concentration gradient, and thus enhances enzyme association. Structural probing by in vivo cross-linking, mass spectrometry and protein docking revealed protein-protein interactions in the natural TCA cycle metabolon. Computer simulation demonstrated the formation of an electrostatic channeling upon association-induced surface charge rearrangement. The other aspect of this dissertation is in vitro fabrication of the TCA cycle metabolon mimics by two strategies. Polymeric co-immobilization-induced multi-enzyme aggregation was assessed by acceptor photobleaching FRET imaging. Structure-based design of engineered metabolons was also exploited to construct a recombinant complex featuring natural orientation and enhanced substrate channeling. |
