On the Mechanism of Action at the Acylation Step of the α-Chymotrypsin-catalyzed Hydrolysis of Anilides
1968; Elsevier BV; Volume: 243; Issue: 13 Linguagem: Inglês
10.1016/s0021-9258(19)34198-5
ISSN1083-351X
Autores Tópico(s)Enzyme function and inhibition
ResumoAbstract The hydrolysis of a series of substituted anilides of N-acetyl-l-tyrosine by α-chymotrypsin has been investigated. Values of the kinetic parameters have been obtained in water and deuterium oxide solvent systems. It has been shown that the isotope effect decreases as the basicity of the substrate is increased. pH dependence studies have shown that the rate of hydrolysis, k2, is independent of pH above pH 7. The Michaelis-Menten constant, Km increases at both low and high pH values. The relative basicities of the substrates have been measured by titration of a series of model compounds, substituted acetanilides, in protonated and deuterated glacial acetic acid. In this way, it has been possible to examine quantitatively the relationship between the basicity of the substrate and the corresponding rate of α-chymotrypsin-catalyzed hydrolysis of the substrates. It has been shown that k2 increases with the basicity of the substrate in both water and deuterium oxide systems. This, together with the observation that the hydrogen isotope effect decreases with increasing basicity, suggests that the substrate is protonated before the transition state. The experimental data are consistent with a mechanism involving a pretransition state protonation equilibrium. The limitations of the present approximate treatment are discussed. It appears to be valid for the anilides studied and for peptides, but not for esters or substituted nitroanilides.
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