Spectral and kinetic properties of a cationic peroxidase secreted by cultured peanut cells

Abstract

It is demonstrated that the cationic peroxidase isolated from the growth medium of cultured peanut cells reacts via the same mechanism as other peroxidases, namely conversion of the native enzyme into compound I by reaction with hydrogen peroxide, followed by two reductions by one-electron donors to compound II and then back to the native enzyme. From the pyridine hemochromogen spectrum it is concluded that the prosthetic group of the native enzyme is ferriprotoporphyrin IX. Optical spectra are recorded for (i) the native (ferric) enzyme and its cyanide, azide, fluoride and alkaline forms, (ii) ferrous peroxidase and its cyanide and carbon monoxide complexes, and (iii) compounds I, II, and III. Equilibrium studies show that the ferric cyanide complex has a dissociation constant of 3.0 ± 0.5 μM over the pH range 3–8. The fluoride complex has a dissociation constant which varies from 1.6 μM at pH 4.0 to 28 μM at pH 4.8. Azide has a much lower affinity than fluoride. The alkaline transition occurs with an apparent pKa value of 9.2. Rate constants were recorded for cyanide binding, the alkaline transition, compound I formation, and for the reactions of compound II with a series of substrates. Similarities and differences to horseradish peroxidase are discussed.