Author(s)

Laura Sánchez Zamorano, Nazaret Hidalgo Cuadrado, Patricia Pérez Galende, Manuel G. Roig, Valery L. Shnyrov

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Departamento de Química Física, Facultad de Química, Universidad de Salamanca, Salamanca, Spain.

Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH₄)SO₄ 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 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 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^H₂O₂, K_m^AH₂, k_cat, K_SI^H₂O₂, K_SI^AH₂ and of the microscopic rate constants k₁ and k₃ 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 H₂O₂ 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.

Roystonea regia; Peroxidase; Steady-State Kinetics; Substrate Inhibition; Mechanism-Based Inactivation Kinetics; Hydrogen Peroxide

Zamorano, L. , Cuadrado, N. , Galende, P. , Roig, M. and Shnyrov, V. (2012) Steady-state kinetics of Roystonea regia palm tree peroxidase. Journal of Biophysical Chemistry, 3, 16-28. doi: 10.4236/jbpc.2012.31002.