(69e) Engineering Artificial Metabolons for Substrate Channeling

Enzymes are valuable catalysts in many applications due to their high catalytic activity and substrate specificity. For multi-enzyme systems, limiting conditions may arise including unfavorable equilibrium and unstable intermediates. In order to better control these reactions, we can look to how enzymes evolved. In the cell, most enzymes belong to cascades, where they exist as parts of sequential multi-enzyme pathways. Over the past few decades, highly organized multi-enzyme complexes within these pathways, termed metabolons, have been the focus of intense research in the field of enzymology. In a metabolon, intermediates can be channeled directly from one enzyme to the next without diffusing into the bulk phase through mechanisms including physical tunnels and electrostatic channels connecting active sites. Metabolon formation and substrate channeling phenomena have several advantages over free enzymes including high local metabolite concentrations, promoting the progression of reactions as well as minimizing the cross-talk between competitive pathways. Overall, 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.

Mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) from the TCA cycle have been well-studied for metabolon formation and substrate channeling of the intermediate oxaloacetate (OAA). We examined the structure of a recombinant mMDH/CS metabolon and located a patch of positively charged amino acids connecting the enzyme active sites along which the negatively charged OAA could travel by bounded diffusion. By mutating a single amino acid along this channel, we inhibited metabolon formation and substrate channeling, decreasing the probability of channeling from 0.99 to 0.23.¹ Next, we use several strategies in order to assemble multi-enzyme complexes to mimic nature. These strategies include protein scaffolds to which enzymes are attached by covalent bonds, DNA scaffolds in combination with DNA-binding proteins, and peptide staples genetically fused to one enzyme with binding affinity for the next enzyme. In this work, we compare the substrate channeling in these engineered metabolons to free enzymes and natural recombinant metabolon complexes.

1. Bulutoglu, B.; Garcia, K. E.; Wu, F.; Minteer, S. D.; Banta, S., Direct evidence for metabolon formation and subatrate channeling in recombinant TCA cycle enzymes, submitted.