Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase.

The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E.

Conformational differences between native and recombinant horseradish peroxidase revealed by tritium planigraphy.

Significant conformational differences between native and recombinant horseradish peroxidase have been shown by tritium planigraphy, which includes a method of thermal activation of tritium followed by amino acid analysis of the protein preparation. Comparison of radioactivity distribution among the amino acid residues with the theoretical (calculated) accessibility shows that the recombinant enzyme is characterized by high hydrophobicity and compactness of folding. The protective role of oligosaccharides in native enzyme has been confirmed.

Expression and refolding of tobacco anionic peroxidase from E. coli inclusion bodies.

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain.

Non-enzymatic interaction of reaction products and substrates in peroxidase catalysis.

A quantitative approach for estimation of the non-enzymatic interaction between ammonium 2,2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation product and a poorly oxidized substrate was developed using a system including tobacco peroxidase, a mediator substrate (ABTS), and a second substrate. The approach is based on the establishment of a pseudo-steady-state concentration of the ABTS oxidation product in the course of co-oxidation with a poor substrate. A mathematical description of the experimental curve shape has been proposed to linearize the kinetic data and estimate the rate constant for such non-enzymatic interaction.

Catalytic properties of tryptophanless recombinant horseradish peroxidase.

Heme-containing plant peroxidases (EC 1.11.1.