Investigations of the Chymotrypsin-catalyzed Hydrolysis of Specific Substrates

Enzyme-substrate binding constants, K's, for chymotrypsin and specific amide (N-acetyl-l-tryptophanamide and N-acetyl-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 chymotrypsin-catalyzed 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 mµ. These spectral changes at 290 mµ, observed in all chymotrypsin-catalyzed 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 first 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 K's and the steady state kinetic parameter Km (app) are equivalent. The pH-dependent rate constant (k23) measured in the chymotrypsin-catalyzed hydrolysis of the ester we have studied is shown to pertain to a second intermediate detected in this reaction, an intermediate not seen in the chymotrypsin-catalyzed hydrolysis of the specific substrate amides. The K's values at pH 8.0 for the complexes of α-chymotrypsin with N-acetyl-l-tryptophanamide and N-acetyl-l-phenylalaninamide were found to be 4.7 mm and 29 mm, respectively, in excellent agreement with the steady state kinetic parameter Km (app). In the case of the α-chymotrypsin-catalyzed hydrolysis of N-acetyl-l-tryptophan ethyl ester, data on the pH dependence of k23 was found to be consistent with a pK (app) value of 6.6 for the group of the enzyme controlling the rate, and with a limiting pH-independent value for k23 of 1800 sec-1. The K's value for this ester and α-chymotrypsin at, for instance, pH 5.0, was found to be 2.3 mm, while Km (app) has been reported as ∼0.08 mm. Values of K's and k23, determined in these experiments, together with previously determined values of the steady state kinetic parameter, kcat, allowed a calculation of Km (app). At pH 5.0, this calculated value is ∼0.06 mm, in reasonable agreement with the experimentally observed value.