Ligand binding to the α-subunit of the α2β2 complex of tryptophan synthase induces the αloop6 closure over the α-active site. This conformational change is associated with the formation of a hydrogen bond between αGly181 NH group and βSer178 carbonyl oxygen, a key event for the triggering of intersubunit allosteric signals. Mutation of βSer178 to Pro and αGly181 to Pro, Ala, Phe and Val abolishes the ligand-induced intersubunit communication. Molecular dynamics methods were applied to simulate the conformation of the highly flexible and crystallographically undetectable open state of αloop6 in the wild type and in the α181 mutants. The open conformation of αloop6 is favoured in the wild type enzyme in the absence of α-ligands, and in the α181 mutants both in the presence and absence of bound ligands. A very good correlation was found between the extent of limited tryptic proteolysis and both the hydrogen bond distance between αX181 and βSer178, obtained from the molecular dynamics simulation, and the hydrogen bond strength, evaluated by HINT, an empirical force field that takes into account both enthalpic and entropic contributions. Comparison of the open and closed conformations of αloop6 suggests a pathway for substrate entrance into the α-active site and provides an explanation for the limited catalytic efficiency of the open state.