CATALYTIC PROPERTIES OF PHE41-]HIS MUTANT OF HORSERADISH-PEROXIDASE EXPRESSED IN ESCHERICHIA-COLI

The recombinant horseradish peroxidase and its single-point F41H mutan t have been reactivated from E. coli inclusion bodies. The influence o f the mutation on the heme entrapment, stability and activity of the e nzyme was demonstrated. The catalytic rate constants for H2O2 cleavage and ammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) oxidation decrease by two and one orders of magnitude, respectively. Unlike the wild-type recombinant horseradish peroxidase, the eliminati on of the ABTS oxidation product is not a rate-determining step for th e mutant. The F41H replacement results in significant changes of kinet ics of iodide ion oxidation. The reaction rate is linear to the concen trations of iodide, H2O2, and the enzyme. The results suggest the dire ct interaction of iodide with the porphyrin ring of the heme. The decr ease in ABTS oxidation activity accompanied by retention of activity i n iodide oxidation in the course of low-dosage radiolysis of the F41H mutant is additional evidence of the direct electron transfer from iod ide to the heme, in contrast to ABTS oxidation, in which the electron transfer chain in the protein molecule is involved.