Overlap Concentration and the Effect of Macromolecular Crowding on Citrate Synthase Activity

Although it is well-known that the environment of mitochondria is a densely packed network of macromolecules, the kinetics of the essential metabolic enzyme, citrate synthase, has been studied only under dilute conditions. To understand how this crowded environment impacts the behavior of citrate synthase, Michaelis-Menten kinetics were measured spectrophotometrically in the presence of synthetic crowders as a function of size, concentration, and identity. The largest factor contributing to crowding effects was the overlap concentration (c*), the concentration above which polymers begin to interact. The presence of the crowder dextran decreased the maximum rate of the reaction by ∼20% in the dilute regime (<c*) and 40% in the semidilute regime (>c*) regardless of polymer size. The disparate effects observed from different crowding agents of similar size also reveal the importance of transient interactions from crowding.