Cascade reactions have attracted great attention in the fields of chemical synthesis, biofuel cells, and biosensors due to multiple benefits, including reduced waste generation and minimal purification requirements. They involve a sequence of chemical transformations that take place within a single reactor, where the product of one reaction step, described as an intermediate, becomes the product of the following step. The transport of these intermediates between neighboring active sites often faces the challenge of desorption into the bulk solvent, as well as competition with the side reactions. The efficiency of cascade reactions is therefore limited by intermediate transport. Nature has evolved several substrate channeling strategies to enable the direct transfer of intermediates between adjacent active sites. In this work, molecular dynamics simulations were performed to computationally understand two main mechanisms of substrate channeling: electrostatic channeling and molecular tunneling. Firstly, we studied the electrostatic channeling of glucose-6-phosphate (G6P) on a poly-arginine peptide connecting the sequential enzymes of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH). Metadynamics is used in conjunction with molecular dynamics simulation to quantify the hopping rate of G6P on the bridge. According to lag time calculations observed via a kinetic Monte Carlo model, a poly-arginine bridge is more efficient at channeling G6P compared to the previously studied poly-lysine bridge. A more complex model of electrostatic channeling was then considered, namely the malate dehydrogenase–citrate synthase complex of the citric acid cycle. The negatively charged intermediate oxalacetate (OAA) travels along a positive surface on the enzyme complex. A Markov state model (MSM) identified the dominant pathway and four bottleneck residues. By conducting a hub score analysis and measuring channeling probabilities, we verified that replacing the experimentally determined positive key residue Arg65 with the neutral residue Ala65 led to a 50% reduction in channeling probability, as observed experimentally. The mechanism of molecular tunneling was studied for an ammonia tunnel in the asparagine synthetase system. Combining molecular dynamics with umbrella sampling, energy profiles were constructed for both the original and the mutant structures with an alanine→ leucine replacement. Due to its larger side chain, leucine caused a narrowing of the tunnel when it replaced alanine in the mutant structure, resulting in the blockage of ammonia, and thus an increase in the local energy profile. Finally, the enzymatic interaction between hexokinase (HK) and glucose-6-phosphate dehydrogenase(G6PDH) was studied with coarse-grained molecular dynamics (CG MD). CG MD simplified the complex system of HK-bridge-G6PDH and allowed the observation of enzymatic configuration change. The relative rotation of G6PDH shows an electrostatic interaction between the enzymes, which is dependent on ionic strength. Overall, this work computationally examines the mechanisms of substrate channeling at an atomic level and acts as a guide to design efficient artificial cascades with substrate channeling.
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