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

Abstract

The inactivation of horseradish peroxidase A2 (HRP-A2) with H2O2 as the sole substrate has been studied. In incubation experiments it was found that the fall in HRP-A2 activity was non-linearly dependent on H2O2 concentrations and that a maximum level of inactivation of approximately 80% (i.e. approximately 20% residual activity) was obtained with 2,000 or more equivalents of H2O2. Further inactivation was only induced at much higher H2O2 concentrations. Spectral changes during incubations of up to 5 days showed the presence of a compound III-like species whose abundance was correlated to the level of resistance observed. Inactivation was pH dependent, the enzyme being much more sensitive under acid conditions. A partition ratio (r1 approximately equals 1,140 at pH 6.5) between inactivation and catalysis was calculated from the data. The kinetics of inactivation followed single exponential time curves and were H2O2 concentration dependent. The apparent maximum rate constant of inactivation was lambdamax=3.56+/-0.07x10(-4)s(-1) and the H2O2 concentration required to give lambdamax/2 was K2=9.94+/-0.52 mM. The relationship lambdamax<ki has been shown to apply and thus the rate constant of inactivation has been calculated as ki=1.9x10(-3)s(-1). HRP-A2 possessed catalase-like oxygen gas-releasing activity, the catalytic constant being k3=2.2 s(-1), and the affinity for H2O2 as K2=23 mM. Catalase-like activity was pH dependent and favoured under more basic conditions. A mechanistic model has been developed and used to explain the behaviour of HRP-A2. The model suggests that, in common with HRP-C, mechanism-based (suicide) inactivation is being observed but that a fraction of the HRP-A2 is protected from inactivation in the form of a modified compound III species.

Effects of single mutations on the stability of horseradish peroxidase to hydrogen peroxide

“…Arnolds and Lin's [9] HRP was expressed in Saccharomyces cerevisiae; perhaps the associated glycosylation aids H 2 O 2 stability [12]. K232N, K241F and T110V yielded a significant increase in H 2 O 2 C 50 using 30 min incubations.…”

“…Hiner and co-workers [12] defined the 'partition ratio' as a mechanism for comparing H 2 O 2 stability of HRP variants over four-hour incubation times at room temperature. This ratio yields the number of H 2 O 2 turnovers occuring prior to enzyme inactivation.…”

Oxidative Processes and Antioxidative Metaloenzymes

“…The first stage of oxidation is the emergence of alkylthiolate (RS -) in the presence of the hydroxyl anion (HO -) (reaction 44). Thiolate reacts with oxygen and produces a thyil radical (RS • ) (reaction 45) [85,94]. The second stage is the reactions with thiols and their emerging radicals (reaction 46, 47).…”

“…In response to the reaction of protein thiols (PrS) with R-O-OH, atoms of sulfur are simultaneously oxidized (frequently those in Cys), forming corresponding monoxides (thiosulfinates) and, where appropriate, further oxidized products containing 2 sulfoxide groups (disulfoxide), sulfone moiety (dioxide, thiosulfonate), sulfoxide and sulfone miety (sulfoxido sulfone, trioxides), and 2 sulfone groups (disulfonates, tetraoxides) [181]. Reactions with hydro and hydrogen peroxides convert thiol proteins also into sulfenic acids (RSOH), which can be further oxidized to higher oxidation states such as sulfinic (RSO2H) and sulfonic (RSO3H) acids [85,169]. Oxidative modifications of critical amino acids within the functional domain of proteins may also occur by S-glutathionylation.…”

“…The redox properties of adduct radicals differ; C5-OH adduct radicals are reducing while C6-OH adduct radicals are oxidizing [161]. In the absence of O2, the oxidation of C5-OH adduct radicals, followed by addition of HO -(or addition of water followed by deprotonation), leads to cytosine glycol and thymine glycol [25,85]. C5-OH-6-peroxyl radicals are formed by addition of O2 to C5-OH adduct radicals at diffusion-controlled rates.…”