WILD-TYPE AND MUTANT FORMS OF RECOMBINANT HORSERADISH-PEROXIDASE-C EXPRESSED IN ESCHERICHIA-COLI - SUBSTRATE SPECIFICITY AND STABILITY UNDER IRRADIATION

Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41 --> His and Phe143 --> Glu, were compared t o the wild-type recombinant enzyme expressed in Escherichia coli in te rms of the enzymatic activity and stability under irradiation. Both mu tations caused a significant decrease in activity, but it was still po ssible to follow the effect of mutations on the key steps of the react ion mechanism. Phe41 can be considered a nonpolar barrier separating h istidine residues in the active center and providing a firm noncovalen t binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The P he143 --> Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor subs trates and free radicals. The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2'-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaia col, and o-phenylene diamine. The study of kinetics and inactivation i s in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less acc essible than that for o-phenylene diamine.