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Membraneless Compartmentalization Facilitates Enzymatic Cascade Reactions and Reduces Substrate Inhibition.

ABSTRACT: Living cells possess membraneless organelles formed by liquid-liquid phase separation. With the aim of better understanding the general functions of membraneless microcompartments, this paper constructs acellular multicompartment reaction systems using an aqueous multiphase system. Membraneless coacervate droplets are placed within a molecularly crowded environment, where a larger dextran (DEX) droplet is submerged in a polyethylene glycol (PEG) solution. The coacervate droplets are capable of sequestering reagents and enzymes with a long retention time, and demonstrate multistep cascading reactions through the liquid-liquid interfaces. The ability to change phase dynamics is also demonstrated through salt-mediated dissolution of coacervate droplets, which leads to the release and mixing of separately sequestered reagents and enzymes. Finally, as phase-separated materials in membraneless organelles are often substrates and substrate analogues for the enzymes sequestered or excluded in the organelles, this paper explores the interaction between DEX and dextranase, an enzyme that hydrolyzes DEX. The results reveal that dextranase suffers from substrate inhibition when partitioned directly in a DEX phase but that this inhibition can be mitigated and reactions greatly accelerated by compartmentalization of dextranase inside a coacervate droplet that is adjacent to, but phase-separated from, the DEX phase. The insight that compartmentalization of enzymes can accelerate reactions by mitigating substrate inhibition is particularly novel and is an example where artificial membraneless organelle-like systems may provide new insights into physiological cell functions.

SUBMITTER: Kojima T

PROVIDER: S-EPMC6258206 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Project description:A growing amount of evidence over the last two decades points to the fact that many enzymes exhibit fluctuations in their catalytic activity, which are associated with conformational changes on a broad range of timescales. The experimental study of this phenomenon, termed dynamic disorder, has become possible thanks to advances in single-molecule enzymology measurement techniques, through which the catalytic activity of individual enzyme molecules can be tracked in time. The biological role and importance of these fluctuations in a system with a small number of enzymes, such as a living cell, have only recently started being explored. In this work, we examine a simple stochastic reaction system consisting of an inflowing substrate and an enzyme with a randomly fluctuating catalytic reaction rate that converts the substrate into an outflowing product. To describe analytically the effect of rate fluctuations on the average substrate abundance at steady state, we derive an explicit formula that connects the relative speed of enzymatic fluctuations with the mean substrate level. Under fairly general modelling assumptions, we demonstrate that the relative speed of rate fluctuations can have a dramatic effect on the mean substrate, and lead to large positive deviations from predictions based on the assumption of deterministic enzyme activity. Our results also establish an interesting connection between the amplification effect and the mixing properties of the Markov process describing the enzymatic activity fluctuations, which can be used to easily predict the fluctuation speed above which such deviations become negligible. As the techniques of single-molecule enzymology continuously evolve, it may soon be possible to study the stochastic phenomena due to enzymatic activity fluctuations within living cells. Our work can be used to formulate experimentally testable hypotheses regarding the nature and magnitude of these fluctuations, as well as their phenotypic consequences.

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Membraneless Compartmentalization Facilitates Enzymatic Cascade Reactions and Reduces Substrate Inhibition.

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