Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH 4 )SO 4 precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, TSK-Gel DEAE-5PW and Superdex-200. The novel peroxidase was characterized as having a molecular weight of 48.2 ± 3.0 kDa and an isoelectric point pI 5.4 ± 0.1. The enzyme forms dimers in solution with approximate molecular weight of 92 ± 2 kDa. Here we investigated the steady-state kinetic mechanism of the H 2 O 2 -supported oxidation of different organic substrates by RPTP. The results of the analysis of the initial rates vs. H 2 O 2 and reducing substrate concentrations were seen to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach used expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters K m H 2 O 2 , K m AH 2 , k cat , K SI H 2 O 2 , K SI AH 2 and of the microscopic rate constants k 1 and k 3 of the shared three-step peroxidase catalytic cycle. Furthermore, the concentration and time-dependences and the mechanism of the suicide inactivation of RPTP by hydrogen peroxide were studied kinetically with guaiacol as co-substrate. The turnover number (r) of H 2 O 2 required to complete the inactivation of the enzyme was 2154 ± 100 and the apparent rate constants of catalysis 185 s –1 and 18 s –1 .
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