Effect of a negative charge on the screening of the active site of horseradish peroxidase

Abstract

The F143E mutant form of the recombinant horseradish peroxidase was reactivated fromE. coli inclusion bodies. The mutation inhibits heme entrapment and results in a decrease in the catalytic activity, mainly affecting the stage of the oxidation of a donor substrate (ABTS, iodide). An increase in stability of the mutant form obtained under radiation inactivation over that of the wild-type recombinant enzyme was observed. The data obtained confirms the proposed location of Phel43 at the entrance of the active center, hence its replacement by the negatively charged glutamic acid residue retards heme entrapment and substrate binding, thus protecting the active center of the enzyme against the radicals generated by radiolysis.