Compétition nucléophile dans les réactions d'hydrolyse enzymatique
Authors: Seydoux F. , Yon J. 
Published: March 2005

The effect of added nucleophiles on the hydrolysis reactions catalyzed by enzymes and proceeding via a covalent intermediate, the acylenzyme, could be interpreted in terms of competitive partitioning of the intermediate between water and nucleophilic reagent. The kinetic parameters should be differently affected according as the acylation or the deacylation is the limiting process. The nucleophilic competition is kinetically analysed and developed as a method for evaluating the kinetic rate constants of the elementary steps. A general scheme involving special binding sites of the enzyme for water and for nucleophilic analogs is proposed. Therefore, the apparent reactivity of the nucleophilic compounds compared to that of water is dependant not only on their intrinsic reactivity, but also on their relative affinities for the binding sites. It has been shown that the experimentally observed lack of saturating effect of the nucleophile is not in desagreement with the existence of such sites, if they are not independant: the water generally in excess compared to the nucleophile saturates its receptor site. The equations obtained according to this assumption are comparable with those derived by Bender from a kinetic scheme discussed in great detail in the present paper. However, when taking into account the existence of binding sites, it is necessary to use as a kinetic variable, the ratio [N]/[W], corresponding to the ratio of the nucleophilic reagent concentration to the water concentration, and not [N] only as in Bender's scheme. The significance of the experimental rate constants is also Werent since it involves the affinity of the binding site for the nucleophilic compound.

The choice of a nucleophilic reagent competing with water is discussed in relation to the possibility of denaturation effects and conformational changes introduced by the variation of the dielectric constant of the medium. The occurrence of such phenomena could interfere with the observation of the nucleophilic competition.

The effect of some alcohols on the tryptic hydrolysis of the following ester substrates: α‐N‐ benzoyl‐l‐arginine ethyl ester, α‐N‐toluene‐sulfonyl‐l‐arginine methyl ester, N‐acetyl‐l‐phenyl‐ alanine methyl ester, l‐lysine methyl and ethyl esters, is experimentally studied as an application. For α‐N‐benzoyl‐l‐arginine ethyl ester and α‐N‐toluene‐sulfonyl‐l‐arginine methyl ester in presence of methanol, kcat, remains constant, but Km greatly increases. The increase of Km arises both from a change of the dielectric constant and from the nucleophilic competition. The tryptic hydrolysis of N‐acetyl‐l‐phenylalanine methyl ester is inhibited by the presence of methanol. For this substrate the k4/k′a ratio corresponding to the deacylation rate constants by methanol and by water, is equal to 1.76. With l‐lysine methyl and ethyl esters in presence of methanol and ethanol, both parameters Km, and kcat2 are modified. For these substrates, the acylation and deacylation rate constants are of the same order of magnitude. The k4/ka′ ratio has been found be equal to 2.05 for both substrates at pH 4.8, 25°, CaCl2 0.025 M. The kinetic parameters have been determined; k3′, the deacylation constant is (25.3 ± 2.1) sec−1 for both substrates, k2 is (108 ± 10) sec−1 for l‐lysine methyl ester and (55.5 ± 5.6) sec−1 for l‐lysine ethyl ester; K3, is 0.14 M and 0.15 M for l‐lysine methyl and ethyl esters respectively. These kinetic parameters have been compared with the corresponding ones for α‐N‐benzoyl‐l‐argine ethyl ester and α‐N‐toluene‐sulfonyl‐l‐arginine methyl ester. The presence of an α‐NH3+ group on the substrate strongly affects the acylation step but remains quite ineffective on the deacylation one. The free energy corresponding to the transformation of the Michaelis complex into the acylenz yme is about −0.45 kcal/mole for l‐lysine methyl ester and −0.6 kcal/mole for l‐lysine ethyl ester. These values are compatible with the transfer of the acyl part of the substrate to the hydroxyl a serine residue.

In all cases, the variation of the experimental kinetic parameters with the [N]/[W] ratio and the independance of the deacylation rate constant of the water concentration [W], suggest the existence of a binding site for water and analogues in the acylenzyme. The generality for the existence of such a site in other hydrolytic enzymes is discussed.