Mechanism-based suicide inactivation of white Spanish broom (Cytisus multiflorus) peroxidase by excess hydrogen peroxide.

Suicide inactivation is a common mechanism observed for haem peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by intermediate highly oxidizing enzyme forms during the catalytic cycle. The time-dependence and the inactivation mechanism of Cytisus multiflorus peroxidase (CMP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferulic acid, guaiacol and o -dianisidine). Catalytic activity decreased following the sequence ABTS > guaiacol > ferulic acid > o -dianisidine. Once the intermediate complex (compound III-H 2 O 2 ) had been formed, competition was established between the catalytic pathway and the suicide inactivation pathway. One mole of CMP afforded around 3790 turnovers of H 2 O 2 for ABTS before its complete inactivation. These results suggest that CMP follows a suicide mechanism, the enzyme not being protected in this case. The mechanism of suicide inactivation is discussed with a view to establishing a broad knowledge base for future rational protein engineering. [ABSTRACT FROM AUTHOR]