Substrate Channeling in an Artificial Metabolon: A Molecular Dynamics Blueprint for an Experimental Peptide Bridge

Abstract

Natural enzyme cascades utilize electrostatic guidance as an effective technique to control the diffusion of charged reaction intermediates between catalytic active sites in a process known as substrate channeling. However, the limited understanding of channeling mechanisms has abated the application of this technique in artificial catalytic cascades. In this work, we utilize molecular dynamics simulations to describe the transport of anionic intermediates (e.g., oxalate and glucose-6-phosphate) on a theoretical cationic α-helix peptide bridge and identify rules for molecular-level design of electrostatic channeling. These simulations allowed us to elucidate a surface diffusion mechanism whereby the anionic intermediate undergoes discrete hydrogen-bonding interactions along adjacent cationic residues on the peptide bridge. Using MD simulations as a foundational blueprint, we synthesized an enzyme complex using a poly­(lysine) peptide chain as a cationic bridge between glucose-6-phosphate dehydrogenase and hexokinase. Stopped-flow lag time experiments demonstrate the ability of the artificially linked enzyme complex to facilitate electrostatic substrate channeling, while an analogous neutral poly­(glycine)-bridged complex was used as a control to isolate proximity effects from artificial substrate channeling.

Spatial Positioning and Chemical Coupling in Coacervate‐in‐Proteinosome Protocells

“…We attributed the threefold kinetic enhancement to the closer spatial and diffusive coupling of the cascade reaction at the HRP‐containing membrane such that H 2 O 2 and ABTS were more effectively channeled between the two enzymes in the early stages of the reaction. In contrast, the reaction rates became comparable at later time stages (Figure m) as diffusive mixing became predominant, in agreement with previous studies on enzyme channelling …”

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“…A major advantage of this approach is that the scaffold architecture, and therefore, the enzyme stoichiometric ratios can be easily controlled in a modular fashion (Pröschel et al ). In a recent example, artificial peptide scaffolded, synthetic metabolons of hexokinase (HXK) and glucose‐6‐phosphate dehydrogenase (G6PDH), were proven to be more efficient than the free enzymes for metabolic engineering (Liu et al ). Such studies suggest that synthetically varying the proportion of TCA cycle enzymes that are complexed, as opposed to unbound, likely represents an interesting strategy for future studies.…”

Quantum Dots and Gold Nanoparticles as Scaffolds for Enzymatic Enhancement: Recent Advances and the Influence of Nanoparticle Size

“…The authors found that this was due to increased enzyme stability and a local environment affect (due to DNA surface affinity or a hydration layer) and not substrate channeling [47]. Another illustrative example is by Liu et al, who used a predominantly-lysine cationic bridge between two coupled enzymes (hexokinase and glucose-6-phosphate dehydrogenase) to facilitate substrate channeling of the dual-negatively charged intermediate [75]. A similarly-long neutral bridge did not show evidence of substrate channeling.…”