The binding of inhibitors to α-chymotrypsin

Abstract

The binding of 3 competitive inhibitors, N-acetyl-D-tryptophan, N-acetyl-L-tryptophan and N-acetyl-D-tryptophan amide, to a-chymotrypsin was studied over the pH range 2.20-9.65 by the technique of equilibrium dialysis. Within the limits of the experimental method, the binding of the uncharged amide inhibitor is independent of pH over the range investigated. The binding of each of the enantiomeric acids is dependent on the ionization of a group on the free enzyme, of apparent pK[alpha] 7.3. It is shown that the ionizing group results in the active site of the enzyme developing a net negative charge above pH 7.3. The enzyme groups responsible are tentatively identified, and the significance of the binding constants with respect to the enzymic catalysis is discussed.