Deuterium isotope effect on the suicide inactivation of tyrosinase in its action on o-diphenols.

A solvent deuterium isotope effect on the inactivation suicide of tyrosinase in its action on o-diphenols (catechol, 4-methylcatechol, and 4-tert-butylcatechol) was observed. This isotope effect, observed during kinetic studies in the transition phase, was higher than that described previously in the steady state, indicating that there is an additional slow step in the suicide inactivation mechanism, which we believe to be responsible for the inactivation. In a proton inventory study of oxidation of o-diphenols, the representation of λmax(D,fn)/λmax(D,f0) versus n (atom fractions of deuterium), where λmax(D,fn) is the maximum apparent inactivation constant for a molar fraction of deuterium (n) and λmax(D,f0) is the corresponding kinetic parameter in a water solution, was linear for all substrates. This suggests that only one of the protons transferred from the two hydroxyl groups of the substrate, which are oxidized in one turnover, is responsible for the isotope effects. We propose that this proton could be the proton transferred from the hydroxyl group of C-2 to the hydroperoxide of the oxytyrosinase form (Eox ) and that it probably causes enzyme inactivation through the reduction of the Cu(2+) A to Cu(0) and its subsequent release from the active site.

Contribution of the intra- and intermolecular routes in autocatalytic zymogen activation: application to pepsinogen activation.

Taking as the starting point a recently suggested reaction scheme for zymogen activation involving intra- and intermolecular routes and the enzyme-zymogen complex, we carry out a complete analysis of the relative contribution of both routes in the process. This analysis suggests the definition of new dimensionless parameters allowing the elaboration, from the values of the rate constants and initial conditions, of the time course of the contribution of the two routes. The procedure mentioned above related to a concrete reaction scheme is extrapolated to any other model of autocatalytic zymogen activation involving intra- and intermolecular routes. Finally, we discuss the contribution of both of the activating routes in pepsinogen activation into pepsin using the values of the kinetic parameters given in the literature.