Participation of an Acidic Group in the Chymotrypsin Catalysis

Abstract

Derivatives of N-acetyl-L-tyrosine anilide with substituents, p-CH₃, p-CH₃O, m-CH₃O, p-Cl and m-G, on the aniline ring, were synthesized as substrates of chymotrypsin [EC 3. 4. 4. 5]. It was confirmed that the acylation of the enzyme was the rate limiting step for these anilide substrates. It was, therefore, possible to study the electronic effect of the substituents on the catalysis of the acylation. The ρ-σ^- plot indicated participation of an acidic group of chymotrypsin in the proton transfer to the substrate molecule. The deuterium isotope effect was observed to depend on the σ- value of the substituent. Such dependence was found to be compatible with a mechanism in which the proton transfer occurs rapidly before the rate limiting step, but not with a mechanism in which the proton transfer occurs simultaneously with the acylation. The pH profile of the catalysis was studied. The temperature dependence of the rate constant of catalysis revealed that the heat of activation changes with the σ^- value more steeply than the free energy of activation. This steeper change is compensated for by a change in the entropy of activation in the reverse direction.