Triosephosphate isomerase (TPI) is a glycolytic homodimer that catalyzes the reversible interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P). In this study, we employ molecular dynamics (MD) simulations to investigate how molecular crowding and spatial confinement influence TPI configuration and substrate interactions within bacterial microcompartments (BMCs). To assess the effects of molecular crowding, we performed simulations with a single TPI dimer and five dimers (concentration = 300 g/L) in a water box, in the presence of multiple DHAP 2- and G3P 2- molecules. Our preliminary results indicate that crowding does not significantly alter the compactness of TPI but enhances substrate interactions near the active site. Additionally, we observe that crowding reduces the time TPI spends in the open conformation of catalytic loop 6, suggesting altered dynamics that may impact catalytic efficiency. To further explore the role of spatial confinement, we are extending our simulations to model TPI encapsulated within the protein shell of the Halangium ochraceum BMC. These models incorporate the SpyCatcher-SpyTag system to mimic enzyme-shell conjugation. BMCs are selectively permeable, protein-based organelles that compartmentalize metabolic enzymes. This work provides foundational insights into how crowded and confined environments affect enzyme structure and dynamics, offering guidance for the rational design of BMC-based nanoreactors for metabolic engineering applications.
Chakraborti et al. (Sun,) studied this question.