Substrate-driven chemotactic assembly in an enzyme cascade

Abstract

Enzymatic catalysis is essential to cell survival. In many instances, enzymes that participate in reaction cascades have been shown to assemble into metabolons in response to the presence of the substrate for the first enzyme. However, what triggers metabolon formation has remained an open question. Through a combination of theory and experiments, we show that enzymes in a cascade can assemble via chemotaxis. We apply microfluidic and fluorescent spectroscopy techniques to study the coordinated movement of the first four enzymes of the glycolysis cascade: hexokinase, phosphoglucose isomerase, phosphofructokinase and aldolase. We show that each enzyme independently follows its own specific substrate gradient, which in turn is produced by the preceding enzymatic reaction. Furthermore, we find that the chemotactic assembly of enzymes occurs even under cytosolic crowding conditions.

Printing Life‐Inspired Subcellular Scale Compartments with Autonomous Molecularly Crowded Confinement

“…Notably, heterogeneous distribution of products in enzymatic reactions, highlighted in these experiments, has previously been observed in other enzymatic systems. Recently, Zhao et al observed spots at similar sizes in microfluidic‐based platforms, due to glycolysis cascade enzyme chemotactic assemblies induced by their specific substrates in crowded conditions . In our case, the localization of CHC in spots can be the result of crowding in environments with limited water content, i.e., a condition where protein–protein interactions have been observed triggering phase separation in intracellular environments .…”

“…In our case, the localization of CHC in spots can be the result of crowding in environments with limited water content, i.e., a condition where protein–protein interactions have been observed triggering phase separation in intracellular environments . On the other side, at high EOMCC concentrations, the more uniform CHC signal from the annuli would be consistent with the lateral growth of the CHC‐rich zones as well as with a lower probability to generate chemotactic‐driven enzymatic assembly . The CYP2E1‐catalyzed reaction in highly concentrated regions (annuli and/or spots) may be interestingly related to its different reactivity in mitochondria and in endoplasmic reticula .…”

Engineering of Metabolic Pathways Using Synthetic Enzyme Complexes

“…Proteinprotein interactions are driven by several mechanisms not covered here (Williamson, 2012). A fresh suggestion is that enzymes show chemotactic movement along their substrate gradient, an effort that could drive their colocalization (Wu et al, 2015;Illien et al, 2017;Agudo-Canalejo et al, 2018;Zhao et al, 2018). These experiments use fluorophore-tagged enzymes to follow movement in microfluidic devices.…”

A Thermodynamic Limit on the Role of Self-Propulsion in Enhanced Enzyme Diffusion

“…The apparent diffusion coefficients of various enzymes, as measured typically by fluorescence correlation spectroscopy, have been observed to increase in the presence of substrate. Examples include FoF1-ATP synthase [1], T7 RNA polymerase [2], T4 DNA polymerase [3], bovine catalase [4,5], jack bean urease [6,4,5], hexokinase [7], fructose biophosphatase aldolase [8,7], alkaline phosphatase [5] and acetylcholinesterase [9]. However, the mechanisms underlying these observations remain largely unexplained.…”