In the preceding paper,(1) we reported on the discovery of potent, nonpeptide inhibitors of the matrix metalloproteinase stromelysin that were prepared by linking two ligands which bind weakly to adjacent sites on the protein. Here we describe the enthalpic and entropic contributions to the observed binding energy for both the linked and unlinked compounds using isothermal titration calorimetry. The results of the calorimetric experiments were interpreted on the basis of NMR-derived structures of stromelysin/inhibitor complexes. In addition, enzyme kinetic assays were performed to measure the cooperative binding of the untethered ligands. For the untethered compounds, the presence of acetohydroxamic acid increases the binding energy of biaryl ligands by similar to 1.3 kcal/mol. This gain in energy is enthalpic in nature and can be attributed, in part, to a direct dispersion interaction between the two ligands. For the linked compounds, enthalpic contributions to the binding energy depend critically on the linker length, whereas the entropic contributions show virtually no dependence. The significant gains in enthalpy observed for a compound which linked the hydroxamate to the biaryl with a two methylene bridge was not observed for compounds with longer linkers due to a difference in the position of the biaryl moiety in the binding pocket. This difference disrupts key interactions between the ligand and the protein and highlights the importance of the linker in the design of tethered compounds.