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Physical and chemical studies of chymotrypsin in solution and kinetic measurements of individual steps in reactions catalysed by this hydrolytic enzyme are reported. The solution studies also provide a basis for correlation of individual reaction steps with structural information obtained by Drs Blow and Hartley and discussed at this Discussion. The rate and equilibrium constants pertaining to the three-step mechanism shown below have been determined for the chymotrypsin catalysed hydrolysis of three specific ester substrates—the ethyl esters of V-acetyl-L-tyrosine, N -acetyl-L-tryptophan, and N -acetyl-L-phenylalanine—at selected pH values with use of both flow and relaxation techniques. iV-acetyl-L-phenylalanine—at selected pH values with use of both flow and relaxation techniques. / * E + S ^ 4 E S -y » EP 2 ,---► E + P 2 , * 2 3 £ 3 4 where E is enzyme, S is substrate, ES is a complex, EP 2 is an intermediate compound, and the products P 1 and P 2 are, respectively, an alcohol or amine and an acid. The hydrolysis of chymotrypsin specific substrates is shown to be considerably more complex than is indicated by this previously proposed mechanism. In addition to the steps shown, there exist a number of ionization and conformational equilibria which account for the bell-shaped pH-rate profiles observed for chymotrypsin catalysed reactions. A catalytically active enzyme conformation, which is in pH dependent equilibrium with a catalytically inactive conformation, is shown to have its origin in the enzyme activation mechanism. In the chymotrypsin catalysed hydrolysis of an amide substrate, furylacryloyltryptophanamide, there has been detected an additional intermediate which differs from the intermediate EP 2 observed in ester hydrolysis.

Hydrogen ion uptake by chymotrypsin during reversible binding of specific substrate is shown to be due to an ionizing group of the enzyme with a pK(apparent) approximately 9 in the free enzyme. This pK(apparent) is shifted to higher value in the enzyme-substrate complexes. Previous results indicating an equilibrium, controlled by this ionizing group, between active and inactive conformational forms of chymotrypsin are confirmed.

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