Hydrolyse trypsique de dérivés d'amino‐acides non substitués en α

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Abstract
The tryptic hydrolysis of l‐lysine methyl ester, l‐lysine paranitroanilide and l‐tyrosine methyl ester has been studied as a function of pH.In addition, these reactions have been studied in the presence of methanol as a nucleophilic reagent wich competes with water in the deacylation step.With lysine methyl ester, a bell‐shaped profile for Kcat vs pH has been observed. The optimum pH for this substrate occurs at pH 5.8. Km decreases 200‐fold when the pH is increased from pH 5 to 8.5. From the nucleophilic competition data, it can be deduced that the k2/k′3 ratio increases between pH 4.8 and 5.8 with deacylation as rate‐determining step.With tyrosine methyl ester, the profiles of both kcat and Km vs pH are bell‐shaped, but the pH optimum in this case occurs at a pH higher than with lysine methyl ester (pH 7.5). The nucleophilic competition data indicate that the acylation step is rate‐limiting for this substrate.With lysine paranitroanilide, kcat increases when the pH is increased from 5 to 6.5, but at higher pH, the kinetic behavior of this substrate is complicated by a non‐Michaelian saturation phenomenon, probably related to transpeptidation.From the quantitative interpretation of our experimental data, the following conclusions could be drawn: The conjugate acids are more reactive than their corresponding α‐amino acid derivatives. In the acylenzyme formed with bicationic conjugate acid of lysine methyl ester, the active imidazole pK is strongly displaced from its normal value to the acidic region. (This pK is lowered from 6.90 in the free enzyme to 4.80 in this acylenzyme.)These two first points explain the anomalous value of the pH optimum for this substrate. The pK shift is less pronounced in the Michaelis complex formed with the conjugate acid of tyrosine methyl ester, lysine methyl ester and lysine paranitroanilide. Since this pK shift can be interpreted as resulting from a repulsive electrostatic interaction between the imidazolium group of enzyme and the α‐ammonium group of the substrate, our data suggest a change of the active site geometry (including substrate) after the acylation step. The comparison of the kinetic parameters obtained in this work with those of corresponding α‐N‐acyl derivatives suggests that the α‐peptidic linkage is an important structural factor for the acylation step but not for the deacylation.Finally, the effect of possible transpeptidation reaction on the kinetic data is discussed.

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