In nature, most enzymes belong to cascades, where they exist as parts of sequential multi-enzyme pathways. Over the past couple decades, highly organized multi-enzyme complexes within these pathways, termed metabolons, have been the focus of intense research in the field of enzymology. Within the metabolon, a substrate can be channeled directly from one enzyme to the next without diffusing into the bulk phase through mechanisms including tunnels and electrostatic channels connecting active sites. Metabolon formation and substrate channeling phenomena have several advantages over free enzymes including high local metabolite concentrations, overcoming unfavorable equilibrium of individual enzymes as well as minimizing the cross-talk between competitive pathways. Overall, a better mass transport within the pathway can be achieved, resulting in enhanced kinetics of the metabolic process, allowing the cell to operate at high efficiency. In this work we study metabolon formation and substrate channeling in three enzymes of the TCA cycle, mitochondrial malate dehydrogenase (MDH), citrate synthase (CS) and aconitase (Aco), which have been shown to be involved in supramolecular complex formation. In order to investigate this metabolon through mutational analysis, we expressed recombinant porcine heart MDH, CS and Aco in E. coli. The recombinant enzymes were characterized for their kinetic parameters and metabolon formation. We showed that the recombinant enzymes form a similar conformation as the natural complex in vivo by resolving the three-dimensional structure with chemical cross-linking and mass spectrometry. In this structure we observed a potential electrostatic pathway of positively charged residues between the active sites of MDH and CS. Important residues in this pathway on CS were identified, and we performed site-directed mutagenesis to study their importance to channeling the negatively charged intermediate. One mutation with similar kinetics to the wild type was further investigated for substrate channeling with a transient time analysis of product formation and kinetic analysis in the presence of a competing pathway. The results of these studies indicated further proof for electrostatic substrate channeling. This study provides an important insight into understanding the coupled reaction-transport systems and the metabolon formation in general.

Artificial TCA cycle metabolon: Direct evidence for metabolon formation and substrate channeling

251st National Meeting (2016)

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