Gold Nanoparticle Templating Increases the Catalytic Rate of an Amylase, Maltase, and Glucokinase Multienzyme Cascade through Substrate Channeling Independent of Surface Curvature

Abstract

The templating of enzymes has shown myriad advantages, including increased stability and kinetic rates. Specifically, the use of nanoparticles (NPs) as templates has been shown to increase the kinetic rates over larger macroscale scaffolds, in part by overcoming diffusion limitations. Within the field, there is considerable debate over the parameters that optimize this interaction, e.g., NP size, curvature, and surface ligands. Recently much interest has been seen in the pursuit of complete enzyme cascades on nanoscaffolds, with the increased enzyme proximity providing multiple benefits. In this work we demonstrate that multienzyme cascades can be templated on individual gold NPs. Utilizing a three-enzyme cascade of amylase, maltase, and glucokinase, we found a ∼3-fold enhancement in product formation when all three enzymes were bound to the same NP as compared to when the enzymes were bound separately and then combined as well as freely diffusing in solution. This strongly suggests that the increased kinetics was due to substrate channeling. In addition, we investigated the effect of size and curvature on the kinetics of the cascade by using different-shaped gold NPs. Substrate channeling was observed in small and large gold nanospheres (∼30 and 70 nm diameters, respectively) and gold nanostars with an equivalent surface area. Equivalent enhanced kinetics for the enzyme cascade were observed for all of the tested nanoscaffolds, in contrast with what has been reported for single enzymes on gold NPs where changes in curvature resulted in modified kinetics. The results of this work should allow for optimized design of NPs and enzyme cascades for in vitro biocatalysis.

Self assembling nanoparticle enzyme clusters provide access to substrate channeling in multienzymatic cascades

“…Kinetic descriptors for each enzyme, both on/off NP include maximum velocity ( V max ), the Michaelis constant ( K M ), catalytic rate ( k cat ), enzyme efficiency ( k cat / K M ), and specific activity (SA) are found in Table 2 21 . Kinetic values are termed “apparent” since it is uncertain if enzyme activity when it is displayed on a NP satisfies all classical Michaelis–Menten (MM) assumptions; nevertheless, they still serve as excellent comparators of activity in the different configurations 37 , 43 , 53 , 54 , 74 .…”

“…Genes for α-amylase from B. cereus , alpha-glucosidase from Saccharomyces pombe which we refer to as maltase 43 , 51 , and INVB an invertase from Zymomonas mobilis 127 , were codon optimized for expression in E. coli and synthesized by Genscript (protein sequences are listed in the Supplementary Materials ). Genes were subsequently cloned into the NcoI and XhoI restriction sites in expression vector pET28a (Addgene).…”

Catalytic Kinetics Considerations and Molecular Tools for the Design of Multienzymatic Cascade Nanoreactors

“…[94] Recently, Diaz et al showed that the gold templating enhanced the catalytic rate of amylase, maltase and glucokinase multienzyme cascade by~3-fold as compared to the enzymes bound separately and combined freely in solution. [95] Thus, it was evident that the enhanced enzyme kinetics was achieved through substrate channeling. Importantly, the evaluation of enzyme cascade on Au nanoscaffolds of different size and shape revealed the equivalent enhanced multienzyme kinetics suggesting substrate channeling independent of surface curvature.…”

High-Affinity Points of Interaction on Antibody Allow Synthesis of Stable and Highly Functional Antibody–Gold Nanoparticle Conjugates

“…Protein−gold nanoparticle conjugates are poised to play a critical role in next-generation biomedical technologies. 1−7 Advances in diagnostic testing, 8−15 tissue and cellular imaging, 16−18 photothermal therapy, 19 biocatalysis, 20 and drug delivery 21 have been achieved with the integration of functionalized gold nanoparticles. The overall success and widespread implementation of these nanoparticle-enabled technologies hinges on the effectiveness of the protein− nanoparticle conjugate, specifically the interfacial chemistry to immobilize the surface protein.…”