Role of the reductant substrates on the inactivation of horseradish peroxidase by m‐Chloroperoxybenzoic acid

Abstract

SUMMARYHorseradish peroxidase reacts with H202 and other hydroperoxides to form Compound I, the first active enzymatic form. m-Chloroperoxybenzoic acid, a xenobiotic hydroperoxide, acts as an oxidant substrate of horseradish peroxidase. However, this hydroperoxide is also a powerful inactivator of the enzyme and in this sense is more effective than H202. The coupled reductant substrates used in the peroxidatic reaction protect the enzyme from the inactivating process.A reaction mechanism is proposed with two competitive routes: one catalytic and one inactivating. Using a kinetic approach, the ratio between the hydroperoxide and the reductant substrate appears to be a decisive factor in the catalytic turnover of the enzyme.The role of the reductant substrates in protecting the enzyme, and the physiological and biotechnological implications of this process are discussed.

The inactivation of horseradish peroxidase isoenzyme AZ by hydrogen peroxide: an example of partial resistance due to the formation of a stable enzyme intermediate

“…However, values of r 2 "2 and r 3 "7 (see Eq. A38) may be postulated, if it is assumed that the behaviour of HRP-A2 with the powerful suicide inactivator m-CPBA is similar to that of HRP-C [20,23,24,25]. Preliminary results (not shown) tend to suggest that this is indeed the case.…”

“…To account for these results a kinetic model (see Schemes 1 and 2, and the Appendix) has been developed using as its basis a previous theoretical examination of the kinetics of mechanism-based (suicide) inactivation [28] and our previous work on HRP-C [5, 18,19,20,21,22,23,24,25].…”

“…The pivotal point of partition between protection and inactivation has been identified as the complex between compound I and peroxide [20]. We have previously studied inactivation and protection in detail using HRP-C (wild-type, recombinant and mutants) with H 2 O 2 [5, 19,21,22,23] and m-chloroperoxybenzoic acid (m-CPBA) [20,23,24,25], which is not a substrate for the catalase-like pathway. A kinetic model and mechanism have been developed, supported by transient and steady state kinetic data, to explain the reaction pathways.…”

The Inactivation and Catalytic Pathways of Horseradish Peroxidase with m-Chloroperoxybenzoic Acid

“…However, when conventional reducing substrates such as ABTS or guaiacol are present, the extent of inactivation is decreased since the steadystate level of compound I, which reacts with the hydroperoxide to initiate inactivation, is much lower, i.e. reducing substrate protects the enzyme from inactivation by removing compound I (38,39).…”

Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2

“…Physiologically, the catalase-like activity of peroxidase might seem largely irrelevant because of the presence of reducing substrates that normally take part in peroxidatic activity and protect the enzyme from inactivation [17,53]. However, under conditions of oxidative stress or during the protective H # O # burst of plants in response to pathogenic attack, an elevated level of oxidants compared with reductants can occur.…”