Conformational changes of enzyme complexes are often related to regulating and creating an optimal environment for efficient chemistry. We investigated the synergistic regulation of the tryptophan synthase (TRPS) complex, studied for decades as a model of allosteric regulation and substrate channeling within protein complexes. TRPS is a bifunctional tetrameric alphabetabetaalpha enzyme complex that exhibits cooperative motions of the alpha- and beta-subunits by tightly controlled allosteric interactions. We have delineated the atomically detailed dynamics and conformational changes of TRPS in the absence and presence of substrates using molecular dynamics simulations. The computed energy and entropy associated with the protein motions also offer mechanistic insights into the conformational fluctuations and the ligand binding mechanism. The flexible alpha-L6 loop samples both open and partially closed conformations in the ligand-free state but shifts to fully closed conformations when its substrates are present. The fully closed conformations are induced by favorable protein-ligand interactions but are partly compensated by configurational entropy loss. Considerable local rearrangements exist during ligand binding processes when the system is searching for energy minima. The motion of the region that closes the beta-subunit during catalysis, the COMM domain, couples with the motion of the alpha-subunit, although the fluctuations are smaller than in the flexible loop regions. Because of multiple conformations of ligand-free TRPS in the open and partially closed states, the alpha-L6 loop fluctuations have preferential directionality, which may facilitate the fully closed conformations induced by alpha- and beta-substrates binding to both subunits. Such cooperative and directional motion may be a general feature that contributes to catalysis in many enzyme complexes.