Microenvironmental effects can masquerade as substrate channelling in cascade biocatalysis

Natural cascades frequently use spatial organization to introduce beneficial substrate channeling mechanisms, a strategy that has been widely mimicked in many engineered multienzyme cascades with enhanced catalysis. Enabled by new molecular scaffolds it is now possible to test the effects of spatial organization on cascade kinetics; however, these scaffolds can also alter the microenvironment experienced by the assembled enzymes. We know from decades of enzyme immobilization research that the microenvironment affects enzymatic activity, thus complicating kinetic analysis. Here, we review these effects and discuss examples that exploit the microenvironment to improve single enzyme and cascade catalysis. In doing so, we highlight the challenges in ascribing kinetic enhancements directly to substrate channeling without first determining the effects of the microenvironment.

Automated summary

Natural cascades often use spatial organization to introduce beneficial substrate channeling mechanisms, which has been widely adopted in engineered multienzyme cascades. Utilizing new molecular scaffolds now allows testing the effects of spatial organization on cascade kinetics, but these scaffolds can also change the microenvironment experienced by the assembled enzymes, complicating kinetic analysis.