Technical Abstract: Macromolecular assemblies play critical roles in regulating cellular functions. The cysteine synthase complex (CSC), which is formed by association of serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), functions as a multienzyme complex that responds to changes in intracellular sulfur levels resulting from metabolic stresses in plants and bacteria to act as a sensor and modulator of thiol metabolism. Here we examine the oligomerization and energetics of formation of the soybean CSC using analytical ultracentrifugation, isothermal titration calorimetry, and surface plasmon resonance. Biophysical examination of CSC oligomerization indicates that this macromolecular assembly from soybean consists of a single SAT trimer and three OASS dimers. Under physiological conditions, the stability of the CSC derives from tight binding resulting from rapid association and extremely slow dissociation of OASS in the assembly, as determined by isothermal titration calorimetry and surface plasmon resonance. Addition of multiple OASS dimers to the SAT trimer displays negative cooperativity and requires the C-terminus of SAT for interaction with OASS. Steady-state kinetic analysis shows that the functional consequences of CSC formation include elevated SAT activity and the release of substrate inhibition and feedback inhibition by cysteine, the final product of the pathway. These results suggest a new model for the architecture of this regulatory complex and additional physiological functions for this macromolecular assembly in modulating plant cysteine biosynthesis.