Kinetic Enhancements in DNA–Enzyme Nanostructures Mimic the Sabatier Principle

Abstract

Advances in DNA bionanotechnology have led to the ability to create structures with well-defined chemical and physical features at the nanoscale. Such nanostructures can be used to create spatially organized enzymatic cascades that promote substrate channeling and result in enhanced cascade kinetics. Here, we investigate the effects of substrate−scaffold interactions on the catalytic activity of an enzyme−DNA complex using horseradish peroxidase (HRP) and a nanoscale DNA scaffold with three addressable sites. Kinetic assays with a library of HRP substrates revealed that DNA scaffolding enhances HRP activity in a manner that is analogous to the Sabatier Principle. In this case, the binding of the substrate is to the scaffold and not to the catalyst, but the Sabatier trend holds: weak and strong binding substrates showed no enhancement in kinetics, whereas intermediately bound substrates result in >300% increase in enzyme activity.M etabolic pathways are often organized in multienzyme complexes that promote the efficient transport and processing of substrates along the pathway, resulting in enhanced pathway kinetics and high yields. 1,2 When engineering new and re-engineering existing pathways, such nanoscale organization and the associated kinetic benefits are often lost. A number of recent efforts to reproduce these kinetic benefits in engineered pathways, both in vitro and in vivo, have focused on enzyme colocalization with nucleic acid 3−7 and protein scaffolding, 8−10 where scaffold is defined as a biomolecular structure to which proteins can be attached at specific sites. As a material, DNA can be used to create precisely defined multidimensional shapes with molecular-level control over structural and chemical features. 11 These capabilities can be used to create multienzyme cascades (or polyvalent enzyme displays) with well-defined spatial organization. However, interactions between enzyme substrates and scaffolds, and their potential effectsbeneficial or detrimentalon enzyme kinetics have yet to be explored. Here, we investigate a potentially advantageous (or constraining) aspects of these designs, interactions between substrates and the DNA scaffold.On the basis of well-known interactions between small molecules and DNA (e.g., DNA stains used in electrophoresis and anticancer drugs 12,13 ) and DNA templating of aniline monomers for the enzyme-mediated synthesis of polyaniline nanowires, 14 we reasoned that interactions between DNA scaffolds and enzyme substrates may affect local substrate concentrations and alter the kinetics of enzymes assembled in enzyme-DNA nanostructures. To investigate this possibility, we used a model system of horseradish peroxidase (HRP) assembled on a nanoscale DNA triangle. We elected to use this system because HRP oxidizes a wide range of chemically distinct substrates, including phenol; charged and uncharged phenolics; and colorimetric substrates, such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3,3,5,5-tetramethylbenzidine (TMB) that are commonly use...

Mechanisms of Enhanced Catalysis in Enzyme–DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis

“…Experimental evidence suggests that enhancements are likely the result of one or more mechanisms, including 1) increased substrate (reactant) concentrations in close proximity to the enzymes, 2) controlled chemical environments beneficial to enzyme activity, and 3) substrate channeling by proximity and bounded diffusion in cascade reactions. Our previous work demonstrates enhanced catalysis through attraction of enzyme substrates to active sites by means of engineered substrate–DNA binding interactions . Similar effects were observed in enzyme–DNA nanostructures with engineered NAD(H) cofactor swing arms.…”

Proximity does not contribute to activity enhancement in the glucose oxidase–horseradish peroxidase cascade

“…clearly showed that enhancement occurs even if there are individual enzyme pairs on each scaffold and the enhancement effect is permanent. Lin and Wheeldon pointed out that attractive interactions between a DNA scaffold and the substrate can facilitate the transport of the substrate to the enzyme2930, and the model by Idan and Hess18 suggested that the effect could be long-lasting. However, DNA does not attract the negatively charged substrate ABTS31 and the throughput enhancement is caused by facilitated transport and should disappear as the concentration of the intermediate substrate builds up in the reaction vessel.…”

Recent Advances in Enzyme-Nanostructure Biocatalysts with Enhanced Activity

“…As shown in Figure 2, nanobiocatalysts with increased activity are constructed through insertion of nanoparticles into enzyme molecules [23], immobilization of enzymes on the surface of nanoparticle/nanorod/2-dimensional nanomaterials [27,28,34], encapsulation of enzymes in porous supports [37,39], embedding/enveloping enzymes on/in 3-dimensional nanostructured supports [16,48,80]. Horseradish peroxidase was immobilized on a nanoscale DNA scaffold with three addressable sites [72]. The enhanced enzyme activity (>300%) of this nanobiocatalyst was ascribed to preferential binding of the substrates to the minor groove of the double DNA helix.…”