Kinetics of the trypsin-catalyzed hydrolysis and ethanolysis of l-lysine methyl ester

1. 1. The reaction of l-lysine methyl ester (LME) and trypsin in aqueous ethanol yields in the first place methanol, lysine and l-lysine ethyl ester (LEE). LEE is then acted upon by trypsin to give lysine and ethanol. Hence, kinetics are complex and involve 4 rate constants and 2 equilibrium constants. 2. 2. An attempt was made to determine Michaelis constant (K_m) and catalytic rate constant (K_cat) for the first reaction, i.e. trypsin-catalyzed cleavage of LME, by analyzing the composition of reaction mixtures at early stages in order to have a ratio LEE:LME as small as possible. 3. 3. At pH 6.5 the reaction follows Michaelis kinetics, but at pH 8.5 double reciprocal plots become curved suggesting activation by excess substrate. Apparently the number of substante molecules bound per enzyme molecule increase as dielectric constant decreases. 4. 4. The value of k_eat for the overall reaction increases as alcohol concentration increases. This effect may be ascribed mainly to an enhanced alcoholysis. The ratio of deacylation constants k₄:k₃ (alcohol:water) is also modified by a change of the medium dielectric constant. From the results presented here the suggestion is possible that the principal effect of lowering the dielectric constant is to increase the rate of acylation with respect to that of dissociation of Michaelis complex.