Tryptophanless recombinant horseradish peroxidase: Stability and catalyticproperties

The tryptophanless mutant of horseradish peroxidase, W117F, has been constr ucted and expressed in Escherichia coli. The mutation affects enzyme foldin g and stability. The optimum composition of the refolding medium requires t he presence of ammonium sulfate. The yield of mutant is ca. 8000 U per lite r of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25,000 U per liter and 2000 U/mg for the recombinant wil d-type enzyme). The mutant is more stable in acid media, in the reaction co urse and toward irradiation. The effect of hydrogen peroxide pretreatment o n radiation-induced inactivation of the wild-type and mutant enzyme indirec tly indicates participation of Trp-117 in electron transfer pathways throug h the enzyme molecule. This is in agreement with the steady-state kinetic d ata interpreted in terms of Trp-117 participation in electron transfer with in the Michaelis complex. (C) 1999 Academic Press.