Evaluation of equilibrium constants for the binding of N-acetyl-L-tryptophan to monomeric and dimeric forms of .alpha.-chymotrypsin

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Abstract
The binding of N-acetyl-L-tryptophan to the monomeric and dimeric forms of .alpha.-chymotrypsin in I [ionic strength] = 0.2 acetate-chloride buffer, pH 3.86, has been studied quantitatively. Equilibrium sedimentation studies in the absence of inhibitor yielded a dimerization constant of 3.5 l/g. This value was confirmed by frontal gel chromatography of the enzyme on Bio-Gel P-30, which was also used to establish that N-acetyl-L-tryptophan binds preferentially to monomeric enzyme. From kinetic studies of competitive inhibition with N-acetyl-L-tryptophan ethyl ester as substrate, an equilibrium constant of 1300 M-1 was determined for the binding of N-acetyl-L-tryptophan to monomeric .alpha.-chymotrypsin. An intrinsic binding constant of 250 M-1 for the corresponding interaction with dimeric enzyme was calculated on the basis of these results and binding data obtained with concentrated (18.5 g/l) .alpha.-chymotrypsin. The present results refute earlier claims for exclusive binding of competitive inhibitors to monomer and also those for equivalence of inhibitor binding to monomeric and dimeric forms of .alpha.-chymotrypsin.