Enzyme assembly on nanoparticle scaffolds enhances cofactor recycling and improves coupled reaction kinetics.

Enzyme activity can be many times enhanced in configurations where they are displayed on a nanoparticle (NP) and this same format sometimes even provides access to channeling phenomena within multienzyme cascades. Here, we demonstrate that such enhancement phenomena can be expanded to enzymatic cofactor recycling along with the coupled enzymatic processes that they are associated with. We begin by showing that the efficiency of glucose driven reduction of nicotinamide adenine dinucleotide (NAD+ → NADH) by glucose dehydrogenase (GDH) is enhanced ca. 5-fold when the enzyme is displayed on nanocrystalline semiconductor quantum dots (QDs) which are utilized as prototypical NP materials in our experimental assays. Coupling this enzymatic step with NADH-dependent lactate dehydrogenase (LDH) conversion of lactate to pyruvate also increases the latter's rate by a similar amount when both enzymes were jointly incorporated into self-assembled QD-based nanoclusters. Detailed agarose gel mobility assays and transmission electron microscopy imaging studies confirm that both tetrameric enzymes assemble to and crosslink the QDs into structured nanoclusters via their multiple-pendant terminal (His)6 sequences. Unexpectedly, control experiments utilizing blocking peptides to prevent enzyme-crosslinking of QDs resulted in even further enhancement of individual enzyme on-QD kinetic activity. This activity was also probed revealing that 200-fold excess peptide/QD addition enhanced individual GDH and LDH on-QD kcat a further 2- and 1.5×, respectively, above that seen just by QD display to a maximum of ∼10-fold GDH enhancement. The potential implications for how these enzyme kinetics-enhancing phenomena can be applied to single and multi-enzyme cascaded reactions in the context of cofactor recycling and cell-free synthetic biology are discussed.