Steady-state kinetics of Roystonea regia palm tree peroxidase

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.

DOI: 10.4236/jbpc.2012.31002                

Abstract

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.

Keywords

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

Conflicts of Interest

The authors declare no conflicts of interest.

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