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. ∼ 20% residual activity) was obtained with 2000 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 (r ₁≈1140 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 λ _max=3.56±0.07×10⁻⁴ s⁻¹ and the H₂O₂ concentration required to give λ _max/2 was K _λ=9.94±0.52 mM. The relationship λ _max<k _i has been shown to apply and thus the rate constant of inactivation has been calculated as k _i=1.9×10⁻³ s⁻¹. HRP-A2 possessed catalase-like oxygen gas-releasing activity, the catalytic constant being k ₃=2.2 s⁻¹, and the affinity for H₂O₂ as K ₂=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.