Ligand binding to the alpha-subunit of the alpha2beta2 complex of tryptophan synthase induces the alphaloop6 closure over the alpha-active site. This conformational change is associated with the formation of a hydrogen bond between alphaGly181 NH group and betaSer178 carbonyl oxygen, a key event for the triggering of intersubunit allosteric signals. Mutation of betaSer178 to Pro and alphaGly181 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 alphaloop6 in the wild type and in the alpha181 mutants. The open conformation of alphaloop6 is favoured in the wild type enzyme in the absence of alpha-ligands, and in the alpha181 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 alphaX181 and betaSer178, 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 alphaloop6 suggests a pathway for substrate entrance into the alpha-active site and provides an explanation for the limited catalytic efficiency of the open state.