ИСТИНА

Horseradish peroxidase (HRP) is one of the most studied heme-containing enzymes of the plant peroxidase superfamily and it is the biocatalyst of high practical interest. Therefore, HRP is widely used in different bioanalytical applications and diagnostic kits. It was reported that enzyme activity and stability of HRP in aqueous buffer and organic media can be greatly improved through the technique of chemical modification of lysine residues. However, this approach is non-specific in contrast to the replacement of native heme with the artificial prosthetic group. Experiments on the production of reconstructed horseradish peroxidase with modified heme demonstrated that SHD incorporation into the apoenzyme efficiently proceeds even when bulky substituents are used for the modification of the heme propionate groups. Moreover, reconstituted enzymes had improved catalytic activity, increased thermostability and solvent tolerance. Thus HRP properties can be modulated by variation of substituents introduced into heme. In order to understand the mechanism and effects of changes in the properties of peroxidase with modified heme derivatives, we performed computer simulations of the three-dimensional structures of the apoenzyme-modified heme complexes. The computer simulation results for the structure of HRP with chemically modified heme are in a good agreement with the experimental data. Thus, computer simulations can be used to design experiments for heme modification. Thereby, we have designed several artificial prosthetic groups (APGs). Computer models for the APG--apoenzyme complexes were obtained. Based on results of computer simulations we have selected a few APGs for following chemical synthesis. The experiments on the reconstitution of apo-HRP with APGs are in progress. This work is supported by Russian Foundation for Basic Research (grant #13-04-02013-а).