Enhancing Coupled Enzymatic Activity by Colocalization on Nanoparticle Surfaces: Kinetic Evidence for Directed Channeling of Intermediates.

Multistep enzymatic cascades are becoming more prevalent in industrial settings as engineers strive to synthesize complex products and pharmaceuticals in economical, environmentally friendly ways. Previous work has shown that immobilizing enzymes on nanoparticles can enhance their activity significantly due to localized interfacial effects, and this enhancement remains in place even when that enzyme's activity is coupled to another enzyme that is still freely diffusing. Here, we investigate the effects of displaying two enzymes with coupled catalytic activity directly on the same nanoparticle surface. For this, the well-characterized enzymes pyruvate kinase (PykA) and lactate dehydrogenase (LDH) were utilized as a model system; they jointly convert phosphoenolpyruvate to lactate in two sequential steps as part of downstream glycolysis. The enzymes were expressed with terminal polyhistidine tags to facilitate their conjugation to semiconductor quantum dots (QDs) which were used here as prototypical nanoparticles. Characterization of enzyme coassembly to two different sized QDs showed a propensity to cross-link into nanoclusters consisting of primarily dimers and some trimers. Individual and joint enzyme activity in this format was extensively investigated in direct comparison to control samples lacking the QD scaffolds. We found that QD association enhances LDH activity by >50-fold and its total turnover by at least 41-fold, and that this high activation appears to be largely due to stabilization of its quarternary structure. When both enzymes are simultaneously bound to the QD surfaces, their colocalization leads to >100-fold improvements in the overall rates of coupled activity. Experimental results in conjunction with detailed kinetic simulations provide evidence that this significant improvement in coupled activity is partially attributable to a combination of enhanced enzymatic activity and stabilization of LDH. More importantly, experiments aimed at disrupting channeled processes and further kinetic modeling suggest that the bulk of the performance enhancement arises from intermediary "channeling" between the QD-colocalized enzymes. A full understanding of the underlying processes that give rise to such enhancements from coupled enzymatic activity on nanoparticle scaffolds can provide design criteria for improved biocatalytic applications.

Pursuing the Promise of Enzymatic Enhancement with Nanoparticle Assemblies.

The growing emphasis on green chemistry, renewable resources, synthetic biology, regio-/stereospecific chemical transformations, and nanotechnology for providing new biological products and therapeutics is reinvigorating research into enzymatic catalysis. Although the promise is profound, many complex issues remain to be addressed before this effort will have a significant impact. Prime among these is to combat the degradation of enzymes frequently seen in ex vivo formats following immobilization to stabilize the enzymes for long-term application and to find ways of enhancing their activity. One promising avenue for progress on these issues is via nanoparticle (NP) display, which has been found in a number of cases to enhance enzyme activity while also improving long-term stability. In this feature article, we discuss the phenomenon of enhanced enzymatic activity at NP interfaces with an emphasis on our own work in this area. Important factors such as NP surface chemistry, bioconjugation approaches, and assay formats are first discussed because they can critically affect the observed enhancement. Examples are given of improved performance for enzymes such as phosphotriesterase, alkaline phosphatase, trypsin, horseradish peroxidase, and ß-galactosidase and in configurations with either the enzyme or the substrate attached to the NP. The putative mechanisms that give rise to the performance boost are discussed along with how detailed kinetic modeling can contribute to their understanding. Given the importance of biosensing, we also highlight how this configuration is already making a significant contribution to NP-based enzymatic sensors. Finally, a perspective is provided on how this field may develop and how NP-based enzymatic enhancement can be extended to coupled systems and multienzyme cascades.