Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates III. DETERMINATION OF INDIVIDUAL RATE CONSTANTS AND ENZYME-SUBSTRATE BINDING CONSTANTS FOR SPECIFIC AMIDE AND ESTER SUBSTRATES*

Enzyme-substrate binding constants, K’e, for chymotrypsin and specific amide (N-acetyl-Mryptophanamide and Nacetyl-L-phenylalaninamide) and ester (N-acetyl-L-tryptophan ethyl ester) substrates have been measured by a proflavin-displacement method. Also, the rate constant for the formation of one intermediate in the chymotrypsincatalyzed hydrolysis of the ester has been determined at selected pH values. These constants have not previously been determined. The proflavin-displacement method used to measure the enzyme complex concentration has previously been found to yield results in agreement with those obtained by measuring the spectral changes of the enzyme at 290 mp. These spectral changes at 290 rnp, observed in all chymotrypsincatalyzed reactions studied, have been shown to arise during the reversible formation of an enzyme-substrate complex which precedes the bond-breaking step. New information important to an understanding of chymotrypsin-catalyzed hydrolysis in general has been obtained from this work. It is shown directly, for the Crst time, that in the chymotrypsin-catalyzed hydrolysis of at least two specific amide substrates, the rate-limiting bond-breaking step follows the reversible formation of an enzyme-substrate complex with a dissociation constant K’s, and that Kls and the steady state kinetic parameter K, (app) are equivalent. The pH-dependent rate constant (ks) measured in the chymotrypsin-catalyzed hydrolysis of the ester we have studied is shown to pertain to a second intermediate detected