FORMATION AND PROPERTIES OF DIMERIC RECOMBINANT HORSERADISH-PEROXIDASE IN A SYSTEM OF REVERSED MICELLES

Wild-type recombinant horseradish peroxidase purified and refolded fro m Escherichia coli inclusion bodies has been studied in the system of bis(2-ethylhexyl)sulphosuccinate sodium salt (Aerosol OT)-reversed mic elles in octane. In contrast with native horseradish peroxidase the wi ld-type recombinant enzyme forms dimeric structures as judged by sedim entation analysis. Peroxidase substrates affect the equilibrium betwee n monomeric and dimeric enzyme forms. The dependence of the catalytic activity of recombinant peroxidase on the degree of hydration of the s urfactant exhibits two maxima with pyrogallol, o-phenylene-diamine, gu aiacol and o-dianisidine, with different ratios of activities for the first and second maxima. The differences ir. activities of monomeric a nd dimeric forms of the recombinant horseradish peroxidase provide evi dence for active-site screening in dimeric forms. This has been used t o model a dimeric structure of recombinant horseradish peroxidase with the screened entrance to the active site. In the model structure obta ined, three of eight glycosylation sites were screened. This might exp lain the absence of dimeric structures in native enzyme peroxidase. Th e system of reversed micelles provides, for the first time, evidence f or the formation of dimeric structures by recombinant plant peroxidase with an altered substrate specificity compared with the native enzyme . Thus one can assume that haem-containing peroxidases in general are able to form dimeric structures.