Cascade Kinetics in an Enzyme-Loaded Aqueous Two-Phase System.

Marko Pavlovic,Lukas Zeininger,Jianrui Zhang,Markus Antonietti,Bernhard V K J Schmidt,Alexander Plucinski

doi:10.1021/acs.langmuir.0c00186

Abstract

Macromolecular crowding plays a critical role in the kinetics of enzymatic reactions. Dynamic compartmentalization of biological components in living cells due to liquid–liquid phase separation represents an important cell regulatory mechanism that can increase enzyme concentration locally and influence the diffusion of substrates. In the present study, we probed partitioning of two enzymes (horseradish-peroxidase and urate-oxidase) in a poly(ethylene glycol)–dextran aqueous two-phase system (ATPS) as a function of salt concentration and ion position in the Hofmeister series. Moreover, we investigated enzymatic cascade reactions and their kinetics within the ATPS, which revealed a strong influence of the ion hydration stemming from the background electrolyte on the partitioning coefficients of proteins following the Hofmeister series. As a result, we were able to realize cross-partitioning of two enzymes because of different protein net charges at a chosen pH. Our study reveals a strong dependency of the enzyme activity on the substrate type and crowding agent interaction on the final kinetics of enzymatic reactions in the ATPS and therefore provides substantial implications en route toward dynamic regulation of reactivity in synthetic protocells.

Highlights

The immense complexity of cell cytosol is responsible for a strong discrepancy between “in vitro” and “in vivo”experimental results on enzymatic reaction kinetics
We investigated the kinetics of an enzymatic cascade reaction inside an enzyme-loaded aqueous two-phase system (ATPS) as a function of the substrate type and crowding agent interaction (Scheme 1)
Partitioning coefficients can be tuned by varying molar mass of the polymers in the polymer−polymer ATPS

Summary

■ INTRODUCTION

The immense complexity of cell cytosol is responsible for a strong discrepancy between “in vitro” and “in vivo”. The ability of the PEG-rich and Dex-rich phases as environments for the cascade reaction (oxidation of uric acid followed by an oxidation of the dye i.e., detected spectrophotometrically) was tested and compared to the case of the absence of polymer (Figure 3) In this case, partitioning of the enzymes took place according to the K values from Figure 1. In pursuance of understanding the previously described discrepancies in enzymatic activity of the two phases, we performed several probes investigating reaction rates of cascade reaction in pure PEG or Dex solution at different concentrations without a formation of the ATPS but with the same ionic strength and pH values as in ATPS assays. Similar interactions are not present among complex-branched polysaccharides, such as Dex, and substrates, which ensured high enzyme catalyzed reaction rates

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES