Calorimetry studies of the present systems have shown that preorganizing the ligands using a conformational constraint them can lead to less favorable binding entropies . This result was characterized as paradoxical, because preorganization is typically expected to reduce the entropic penalty for immobilizing a ligand in the binding site . The authors of the experimental paper noted that this paradox would be resolved if the constrained ligands reduce the motions of the binding site residues more than do the flexible ligands. The prior simulation study did not examine this hypothesis, but provided intriguing evidence that the flexible ligands actually are more conformationally restricted in solution than the constrained ones, due to formation of stabilizing nonbonded interactions that the constrained ligands do not access . The present results are consistent with both of these suggestions, as we observe a greater loss of configurational entropy in the binding site for the constrained than the flexible ligands; and we also see that the flexible ligands are less conformationally mobile when free in solution. However, it is the binding site differences that dominate here, with configurational entropy differences of about 5-5.5 kcal/mol, compared with 1.5-3.3 kcal/mol for the ligands. It should be kept in mind that both the prior and the present studies used approximate methods to estimate changes in configurational entropy, and that neither study includes contributions from water or the bulk of the protein. Nonetheless, this is, to our knowledge, the first computational study that addresses the influence of ligand flexibility on the configurational entropy of the binding site.