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

Abstract

H₂O₂ is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H₂O₂ is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H₂O₂ to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H₂O₂ concentration, with values for K₂ (affinity of the first intermediate, compound I, for H₂O₂) and k₃ (apparent rate constant controlling catalase activity) of 4.0±;0.6mM and 1.78±;0.12s^-1 respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H₂O₂. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation.