Enzyme:Nanoparticle Bioconjugates with Two Sequential Enzymes: Stoichiometry and Activity of Malate Dehydrogenase and Citrate Synthase on Au Nanoparticles

Abstract

We report the synthesis and characterization of bioconjugates in which the enzymes malate dehydrogenase (MDH) and/or citrate synthase (CS) were adsorbed to 30 nm diameter Au nanoparticles. Enzyme:Au stoichiometry and kinetic parameters (specific activity, kcat, KM, and activity per particle) were determined for MDH:Au, CS:Au, and three types of dual-activity MDH/CS:Au bioconjugates. For single-activity bioconjugates (MDH:Au and CS:Au), the number of enzyme molecules adsorbed per particle was dependent upon the enzyme concentration in solution, with multilayers forming at high enzyme:Au solution ratios. The specific activity of adsorbed enzyme increased with increasing number adsorbed per particle for CS:Au, but was less sensitive to stoichiometry for MDH:Au. Dual activity bioconjugates were prepared in three ways: (1) by adsorption of MDH followed by CS, (2) by adsorption of CS followed by MDH, and (3) by coadsorption of both enzymes from the same solution. The resulting bioconjugates differed substantially in the number of enzyme molecules adsorbed per particle, the specific activity of the adsorbed enzymes, and also the enzymatic activity per particle. Bioconjugates formed by adding CS to the Au nanoparticles before MDH was added exhibited higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also had 3-fold higher per-particle sequential activity for conversion of malate to citrate, despite substantially fewer copies of both enzymes present.

Graphene‐Oxide‐Based Enzyme Nanoarchitectonics for Substrate Channeling

“…There is also the possibility of nonspecific adsorption between enzymes, which can have a negative effect on the active site conformation, resulting in decreased activity, as observed in this study. Previous studies conducted with simultaneous adsorption of MDH and CS on gold nanoparticles suggested interactions between enzymes in solution before immobilization . With sequential immobilization, there is improved control over the enzyme loading, which can be quantified, and a reduced effect of enzyme folding/unfolding on hydrophobic surfaces by the presence of other enzymes.…”

“…Most of the strategies developed for the study of substrate channeling are based on spatial organization on supports that require complex syntheses. In addition, studies involving the immobilization of malate dehydrogenase (MDH) and citrate synthase (CS) showed that there was decreased specific activity caused by enzyme structural changes . There is a lack of understanding of enzyme structural changes and conformations that can enhance activity and substrate channeling.…”

Systematic Synthesis of ZnO Nanostructures

“…Attachment to a support minimizes unwanted interactions between enzyme molecules. In contrast, a combination of free enzymes in solution can result in unproductive interactions, as has been demonstrated for lipase–trypsin53 and malate dehydrogenase–citrate synthase 54…”

Biomimicry Enhances Sequential Reactions of Tethered Glycolytic Enzymes, TPI and GAPDHS

“…In many cases, these enzymes are embedded to restrict their diffusion [18], [19]; in other cases, the enzymes are truly tethered, usually via chemical attachment strategies. Building upon these advancements, there is growing interest in rebuilding entire biological pathways on hybrid systems [20], [21].…”