A kinetic model for analysis of physical tunnels in sequentially acting enzymes with direct proximity channeling

•Physical tunnel between cascade enzymes is responsible for proximity channeling.•A parameter of kdiff/V0 was used to describe the properties of the physical tunnel.•Proximity channeling is beneficial in pathways with low enzyme activities.•Proximity channeling is more beneficial in pathways with strong side reactions.

Direct channeling is a well-known process in which intermediates are funneled between enzyme active sites through a physical tunnel and can be a potential way to enhance the biocatalytic efficiency for cascading bioreactions. However, the exact mechanism of the substrate channeling remains unclear. In this work, we used mathematical models to describe the mass transfer in the physical tunnels and to gain further understanding of direct proximity channeling. Simulation with a diffusion-reaction model showed that the reduction of the diffusion distance of intermediates could not cause proximity channeling. A second kinetic model, which considered the physical tunnel as a small sphere capable of preventing diffusion of the intermediate into the bulk, was then constructed. It was used to show that the maximum channeling degree in branched pathways depends on the strength of the side reactions, suggesting that proximity channeling in a physical tunnel is more suitable for a pathway with strong side reactions. On the other hand, for a linear pathway, proximity channeling is more beneficial when the constituting enzymes have relatively low activities and expression levels. Our kinetic model provides a theoretical basis for engineering proximity channeling between sequentially acting enzymes in microbial cell factories and enzyme engineering.