Understanding How Nanoparticle Attachment Enhances Phosphotriesterase Kinetic Efficiency

Abstract

As a specific example of the enhancement of enzymatic activity that can be induced by nanoparticles, we investigate the hydrolysis of the organophosphate paraoxon by phosphotriesterase (PTE) when the latter is displayed on semiconductor quantum dots (QDs). PTE conjugation to QDs underwent extensive characterization including structural simulations, electrophoretic mobility shift assays, and dynamic light scattering to confirm orientational and ratiometric control over enzyme display which appears to be necessary for enhancement. PTE hydrolytic activity was then examined when attached to ca. 4 and 9 nm diameter QDs in comparison to controls of freely diffusing enzyme alone. The results confirm that the activity of the QD conjugates significantly exceeded that of freely diffusing PTE in both initial rate (∼4-fold) and enzymatic efficiency (∼2-fold). To probe kinetic acceleration, various modified assays including those with increased temperature, presence of a competitive inhibitor, and increased viscosity were undertaken to measure the activation energy and dissociation rates. Cumulatively, the data indicate that the higher activity is due to an acceleration in enzyme-product dissociation that is presumably driven by the markedly different microenvironment of the PTE-QD bioconjugate's hydration layer. This report highlights how a specific change in an enzymatic mechanism can be both identified and directly linked to its enhanced activity when displayed on a nanoparticle. Moreover, the generality of the mechanism suggests that it could well be responsible for other examples of nanoparticle-enhanced catalysis.

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications

“…177,286 Facilitating this chemistry, laboratory expression and purification of proteins will often have the protein recombinantly modified with a terminal hexahistidine (His 6 ) sequence for IMAC and thus it is prepared in a manner that allows both purification and subsequent coordination to the QD. 120,286,287 Moreover, the small size of these peptidyl tags and its common placement at the protein's termini translates into them having little to no effect on subsequent protein function. Peptides can be easily synthesized to display a terminal His 6 and several ligation chemistries have been developed to modify DNA with such peptidyl motifs.…”

Printed Graphene Electrochemical Biosensors Fabricated by Inkjet Maskless Lithography for Rapid and Sensitive Detection of Organophosphates

“…These enzyme performance characteristics were a slight improvement compared to our previous reports for PTE synthesized in a similar fashion and hence validated that the recombinant protein was properly expressed and incubated. 33,56 The PtNP-IML-PGE was biofunctionalized with PTE enzymes by covalent cross-linking with glutaraldehyde and albumin. Glutaraldehyde cross-linking offers a straightforward and inexpensive approach to immobilizing enzymes onto electrode surfaces and in particular has shown to enhance the biosensor thermostability, reusability, and shelf-life when used to covalently link enzymes to graphene/graphene oxide electrodes.…”

Molecular binding scaffolds increase local substrate concentration enhancing the enzymatic hydrolysis of VX nerve agent

“…Our approach is one of an increasing number of strategies designed to enhance enzyme catalysis by engineering the enzyme microenvironment (Lancaster, Abdallah, Banta, & Wheeldon, 2018). For example, a series of recent studies developed and optimized a PTE‐DNA‐Au nanoparticle system that enhances k cat by altering the reaction mechanism (Breger et al, 2015; Breger et al, 2017; Samanta et al, 2018). PTE‐Protein hydrogel systems also modified reaction kinetics by altering the local chemical environment (Campbell et al, 2018; Lu, Wheeldon, & Banta, 2010).…”