Determination of dissociation constants of crystalline α-chymotrypsin complexes

As a first step in investigations of the properties of crystalline enzymes, the binding of indole, N-formyl- l-phenylalanine, and N-formyl- l- p-iodophenylalanine to α-chymotrypsin crystals, and the binding of indole to tosyl-α-chymotrypsin crystals, has been studied. The methods used were spectrophotometric measurements of the concentration of indole in the supernatant, or measurements of the concentration of radioactively labeled indole in both the supernatant and the crystal. The dissociation constants of the specific binding site of the crystalline enzyme have been determined for indole and N-formyl- l-phenylalanine. It was found that indole does not bind to tosyl-α-chymotrypsin crystals and that N-formyl- p-iodophenylalanine does not bind to the substrate binding site of the crystalline enzyme. The information obtained from these simple equilibrium measurements is in agreement with X-ray diffraction studies. The approach is, therefore, capable of determining whether or not compounds bind to the active site of a crystalline enzyme, and whether the occupancy of this site is sufficient for structure determinations using X-ray diffraction methods.