Large cosolutes, small cosolutes, and dihydrofolate reductase activity

Abstract

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, and 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, falling 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 threefold 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.

Keywords: concentration-dependent crowding; crowding and enzyme activity; enzyme kinetics; macromolecular crowding; size-dependent crowding.

Scheme 1

DHFR reaction scheme. A hydride is transferred from NADPH to dihydrofolate (DHF), forming the reduced product, tetrahydrofolate (THF), and the oxidized cofactor, NADP⁺.

Figure 1

Relative viscosity (A–C) as a function of concentration. (A) Sucrose, black; Ficoll 70, red; Ficoll 400, blue. (B) Glucose, black; dextran 20, red. (C) NEP, black; PVP 10, red; PVP 40, blue; PVP 55, green. Uncertainties for the viscosity measurements are the standard deviation of the mean from triplicate measurements.

Figure 2

DHFR activity as a function of concentration (A–C), volume occupancy (D–F), and relative viscosity (G–I). (A, D, G) Sucrose, black; Ficoll 70, red; Ficoll 400, blue. (B, E, H): Glucose, black; dextran 20, red. (C, F, I) NEP, black; PVP 10, red; PVP 40, blue; PVP 55, green. Asterisks indicate c*, the polymer overlap concentration. Error bars represent standard deviations of the mean from triplicate experiments.