Steady-state kinetics of Roystonea regia palm tree peroxidase

Abstract

Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roys- tonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 pre- cipitation, extraction of palm leaf colored com- pounds and consecutive chromatography on Phenyl-Sepharose, TSK-Gel DEAE-5PW and Su- perdex-200. The novel peroxidase was charac- terized as having a molecular weight of 48.2 ± 3.0 kDa and an isoelectric point pI 5.4 ± 0.1. The en- zyme forms dimers in solution with approximate molecular weight of 92 ± 2 kDa. Here we investi- gated the steady-state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by RPTP. The results of the analysis of the initial rates vs. H2O2 and reducing substrate concentrations were seen to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction me- chanism. 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 Km , m , kcat, SI , SI and of the microscopic rate constants k1 and k3 of the shared three-step peroxidase cata- lytic cycle. Furthermore, the concentration and time-dependences and the mechanism of the sui- cide inactivation of RPTP by hydrogen peroxide were studied kinetically with guaiacol as co-substrate. The turnover number (r) of H2O2 re- quired to complete the inactivation of the enzyme was 2154 ± 100 and the apparent rate constants of catalysis 185 s–1 an –1 2 2 H O K 2 AH K 2 2 H O K 2 AH d 18 s .