Reactivity of Horseradish Peroxidase Compound II toward Substrates:  Kinetic Evidence for a Two-Step Mechanism

Abstract

Transient kinetic analysis of biphasic, single turnover data for the reaction of 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with horseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M(-)(1) s(-)(1)) prior to rate-limiting electron transfer (k(+6) = 42.1 s(-)(1)). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to minimize absorbance changes in the Soret region due to the accumulation of ABTS cation radicals. A steady-state kinetic analysis of the reaction confirmed that the reduction of HRPC compound II by this substrate is rate-limiting in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorbate in the assay medium to minimize the effects of nonenzymic reactions involving phenol-derived radical products. This enabled the initial rates of o-diphenol oxidation at different hydrogen peroxide and o-diphenol concentrations to be determined from the lag period induced by the presence of ascorbate. The kinetic analysis resolved the reaction of HRPC compound II with o-diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphenols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s(-)(1)) is rate-limiting. The size and hydrophobicity of the o-diphenol substrates are correlated with their rate of binding to HRPC, while the electron density at the C-4 hydroxyl group predominantly influences the rate of electron transfer to the heme.

Components of apoplastic ascorbate use in Betula pendula leaves exposed to CO2 and O3 enrichment

“…This suggestion was supported by higher preference of the enzyme toward guaiacol and CA (Table 1). Bimolecular rate constants of the horseradish peroxidase cycle for ASC are shown to be up to 10 2 times lower than for guaiacol (Yamasaki & Yokota, 1973;Rodrígues-López et al, 2000). Thus, our results for birch confirm earlier data for other species that ASC is a poor direct substrate for unspecific PX (Takahama & Oniki, 1992).…”

“…The substrate use rates at physiologically relevant concentrations in vivo were calculated according to the formula for substituted-enzyme catalysis (Rodrígues-López et al, 2000;Cornish-Bowden, 2004). 1.61 ± 0.21 14.00 ± 1.62…”

Absence of a Detectable Intermediate in the Compound I Formation of Horseradish Peroxidase at Ambient Temperature*

“…From our improved estimate of the rate of O-O bond cleavage, we can conclude that Arg-38 plays a more important role in the heterolysis than the assumed role based on the wrong estimate. Furthermore, the previous rate constant (ϳ10 3 s Ϫ1 ) was a basis for considering the O-O bond heterolysis as the rate-limiting step in turnover reactions of HRP with fast substrates (43). Our data show that some other step or steps, such as electron transfer in the compound II-substrate complex (44), are likely to be rate-limiting and not the O-O bond cleavage as previously stated (43).…”

Horseradish Peroxidase-Catalyzed Oxidation of Rifampicin: Reaction Rate Enhancement by Co-oxidation with Anti-inflammatory Drugs

“…1-3 in Chart 1). 15,16) Since the oxidation of rifampicin matches these criteria, we used this model to determine the turnover number (k cat ) for its oxidation.…”