Functionally important carboxyl groups of horseradish peroxidase

Abstract

The carboxyl groups of horseradish peroxidase (EC 1.11.1.7) were modified by the Koshland method using o-dianisidine as a nucleophilic agent ( o-dianisidine, covalently bound to protein, has ϵ 305 = 19.0 M −1·cm −1). At pH 5.0. only four COOH groups were accessible to modification. The modification of three of them decreased the peroxidase activity to the hydrogen donor, o-dianisidine, by 70% but did not alter its activity to the electron donor, potassium ferrocyanide. The constants, k 3 and k 4, of electron transfer from the hydrogen donor to the peroxidase compounds E 1 and E 2, decreased 10- and 1.5-fold, respectively. The modification of the fourth COOH group did not affect the enzyme activity. The hemin isolated from the o-dianisidine-modified peroxidase (M-hemin) had one modified propionate residue. The second propionate was inaccessible to modification. The activity of peroxidase reconstructed from M-hemin and native apoprotein was close to that of the native enzyme. It is concluded that the three functionally important COOH groups are of protein nature and are not directly involved in the enzyme-active site, but important for binding and oxidation of o-dianisidine.