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 to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phe143-->Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates 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), guaiacol, and O-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for O-phenylene diamine.

[Production and catalytic properties of point mutants Phe41--->His and Phe143--->Glu of horseradish peroxidase, expressed in Escherichia coli]. / Poluchenie i kataliticheskie svoistva tochechnykh mutantov Phe41--->His i Phe143--->Glu peroksidazy khrena, ékspressiruemykh v Escherichia coli.

Recombinant horseradish peroxidase and its single-point mutants, F4IH and H143E, have been reactivated from E. coli inclusion bodies with a 25% yield. Both mutations affect heme entrapment as well as enzyme stability and activity. A more than 40-fold decrease in the specific activity towards ABTS is associated with different steps of peroxidase catalysis. F41H replacement results in a drop of both rate constants by two and one orders or magnitude for hydrogen peroxide and ABTS, respectively. The mechanism of iodide oxidation by the F41H mutant fits into a ternary interaction. The F143E replacement mainly affects the steps of ABTS oxidation and product dissociation. It is suggested that the role of replaced phenylalanine residues consists in the formation of a highly hydrophobic pocket allowing for strong non-covalent binding of the heme porphyrin ring.

Effect of single-point mutations Phe41-->His and Phe143-->Glu on folding and catalytic properties of recombinant horseradish peroxidase expressed in E. coli.

Wild-type recombinant and Phe41-->His and Phe143-->Glu mutant forms of horseradish peroxidase have been expressed in E. coli and reactivated from inclusion bodies with a yield of about 25%. The purified homogeneous preparations have been studied in the reaction of ABTS oxidation. The effect of mutations on heme entrapment and kinetics of ABTS oxidation demonstrates the essential role of the replaced residues in providing the hydrophobic crevice for the non-covalent heme binding.