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The catalytic cycle of heme peroxidases involves three processes: the formation of compound I, its conversion to compound II and regeneration of the native enzyme. Each of the processes consists of a reversible binding stage followed by an irreversible transformation stage. Our group has proposed a continuous, sensitive and reliable chronometric method for measuring the steady-state rate of peroxidase activity. Furthermore, we have derived an analytical expression for the steady-state rate and simplified it, taking into consideration the experimental values of the rate constants of some stages previously determined by other authors in stopped-flow assays. We determined the value of the constant for the transformation of a series of phenols and anilines by compound II, and found that it involves a deprotonation step and an electron transfer step. Study of the solvent deuterium isotope effect on the oxidation of phenol revealed the non-rate-limiting character of the deprotonation step in a proton inventory study. Usage of the Marcus equation showed that the electronic transfer step is rate-limiting in both cases, while phenols and anilines were oxidised at different rates for the same potentials. This can be attributed to the shorter electron-tunnelling distance for electron transfer to the iron ion in the phenols than in the anilines. [ABSTRACT FROM AUTHOR]

We report here on the stereospecificity observed in the action of horseradish peroxidase (HRPC) on monophenol and diphenol substrates. Several enantiomers of monophenols and o -diphenols were assayed: L-tyrosinol, D-tyrosinol, L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-a-methyldopa, DL-a-methyldopa, DL-adrenaline, D-adrenaline, L-isoproterenol, DL-isoproterenol and D-isoproterenol. The electronic density at the carbon atoms in the C-1 and C-2 positions of the benzene ring were determined by NMR assays (d 1 and d 2 ). This value is related to the nucleophilic power of the oxygen atom of the hydroxyl groups and to its oxidation-reduction capacity. The spatial orientation of the ring substituents resulted in lower K m values for L- than for D-isomers. The k cat values for substrates capable of saturating the enzyme were lower for D- than for L-isomers, although both have the same d 1 and d 2 NMR values for carbons C-1 and C-2, and therefore the same oxidation-reduction potential. In the case of substrates that cannot saturate the enzyme, the values of the binding constant for compound II (an intermediate in the catalytic cycle) followed the order: L-isomer>DL-isomer>D-isomer. Therefore, horseradish peroxidase showed stereospecificity in its affinity toward its substrates ( K m ) and in their transformation reaction rates ( k cat ). [ABSTRACT FROM AUTHOR]

The kinetic characterization of horseradish peroxidase (HRPC) substrates is difficult because the reaction products are free radicals. The application of a spectropho-tometrical method, which is based on determining the time necessary for a given quantity of L-ascorbic acid to be consumed (lag period) during its reaction with the free radicals generated by the enzyme acting on the reducing substrate, makes it possible to obtain the initial steady-state rates (v0). From the kinetic study of a series of derivates of phenol and aniline, the following parameters were determined for the first time: the global catalytic constant (kcat), the Michaelis constant of HRPC for H2O2 in the presence of each reducing substrate (KH2O2), the Michaelis constant of HRPC for the reducing substrate (Ksm), the binding constant of the reducing substrate with HRPC compound II (k5) and the rate constant of substrate oxidation by HRPC compound II (k6). The values obtained are disccussed. [ABSTRACT FROM AUTHOR]