3 years ago

Large cosolutes, small cosolutes and dihydrofolate reductase activity

Gary J. Pielak, Gerardo M. Perez Goncalves, Annelise H. Gorensek-Benitez, Luis C. Acosta
Protein enzymes are the main catalysts in the crowded and complex cellular interior, but their activity is almost always studied in dilute buffered solutions. Studies that attempt to recreate the cellular interior in vitro often utilize synthetic polymers as crowding agents. Here, we report the effects of the synthetic polymer cosolutes Ficoll, dextran and polyvinylpyrrolidone, as well as their respective monomers, sucrose, glucose and 1-ethyl-2-pyrrolidone, on the activity of the 18-kDa monomeric enzyme, Escherichia coli dihydrofolate reductase. At low concentrations, reductase activity increases relative to buffer and monomers, suggesting a macromolecular effect. However, the effect decreases at higher concentrations, approaching, and, in some cases falls below buffer values. We also assessed activity in terms of volume occupancy, viscosity and the overlap concentration (where polymers form an interwoven mesh). The trends vary with polymer family, but changes in activity are within three-fold of buffer values. We also compiled and analyzed results from previous studies and conclude that alterations of steady-state enzyme kinetics in solutions crowded with synthetic polymers are idiosyncratic with respect to the crowding agent and enzyme. This article is protected by copyright. All rights reserved.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/pro.3316

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