Macromolecular Crowding Effects on Citrate Synthase Kinetics are Concentration‐Dependent

Abstract

Enzyme studies are usually conducted in dilute, in‐vitro conditions; however, enzymes operate within extremely crowded intracellular environments, where macromolecules such as proteins, carbohydrates, and lipids occupy up to 40 % of the available volume. This macromolecular crowding slows diffusion, alters protein structure, and has erratic consequences on enzyme kinetics. To study the potential impacts of crowding on enzyme behavior, Michaelis‐Menten kinetics were employed to monitor citrate synthase reaction rates under crowded conditions. The synthetic polymer dextran was used to mimic the crowded cell interior, and assays were conducted in the presence and absence of dextran at various sizes (10 kDa ‐ 550 kDa) and concentrations (25 g/L ‐ 300 g/L). Crowding decreased the maximum reaction rate, Vmax, for all dextran conditions; however, the severity of this decrease in reaction rate depended on the concentration of dextran. For dextrans at concentrations below their overlap concentrations (c*), crowding resulted in a 10% ‐ 20% decrease in Vmax. However, the reaction rate dropped 30% ‐ 50% at dextran concentrations above c*. The impact of mixtures of dextran sizes on citrate synthase kinetics is also being studied. Taken together, these results indicate that citrate synthase activity is sensitive to the overlap concentration of these dextran solutions.