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The Inactivation and Catalytic Pathways of Horseradish Peroxidase with m-Chloroperoxybenzoic Acid

1997; Elsevier BV; Volume: 272; Issue: 9 Linguagem: Inglês

10.1074/jbc.272.9.5469

1083-351X

José Neptuno Rodríguez‐López, Josefa Hernández‐Ruíz, Francisco Garcı́a-Cánovas, R. N. F. Thorneley, Manuel Acosta, Marino B. Arnao,

Electrochemical sensors and biosensors

The kinetics of the catalytic cycle and irreversible inactivation of horseradish peroxidase C (HRP-C) reacting with m-chloroperoxybenzoic acid (mCPBA) have been studied by conventional and stopped-flow spectrophotometry. mCPBA oxidized HRP-C to compound I with a second order-rate constant k1 = 3.6 × 107M−1 s−1 at pH 7.0, 25°C. Excess mCPBA subsequently acted as a one-electron reducing substrate, converting compound I to compound II and compound II to resting, ferric enzyme. In both of these reactions, spectrally distinct, transient forms of the enzyme were observed (λmax = 411 nm, ϵ = 45 mM−1 cm−1 for compound I with mCPBA, and λmax = 408 nm, ϵ = 77 mM−1 cm−1 for compound II with mCPBA). The compound I-mCPBA intermediate (shown by near infrared spectroscopy to be identical to P965) decayed either to compound II in a catalytic cycle (k3 = 6.4 × 10−3 s−1) or, in a competing inactivation reaction, to verdohemoprotein (ki = 3.3 × 10−3 s−1). Thus, a partition ratio of r = 2 is obtained for the inactivation of ferric HRP-C by mCPBA. The intermediate formed from compound II with mCPBA is not part of the inactivation pathway and only decays via the catalytic cycle to give resting, ferric enzyme (k5 = 1.0 × 10−3 s−1). The data are compared with those from earlier steady-state kinetic studies and demonstrate the importance of single turnover experiments. The results are discussed in terms of the physiologically relevant reactions of plant peroxidases with hydrogen peroxide. The kinetics of the catalytic cycle and irreversible inactivation of horseradish peroxidase C (HRP-C) reacting with m-chloroperoxybenzoic acid (mCPBA) have been studied by conventional and stopped-flow spectrophotometry. mCPBA oxidized HRP-C to compound I with a second order-rate constant k1 = 3.6 × 107M−1 s−1 at pH 7.0, 25°C. Excess mCPBA subsequently acted as a one-electron reducing substrate, converting compound I to compound II and compound II to resting, ferric enzyme. In both of these reactions, spectrally distinct, transient forms of the enzyme were observed (λmax = 411 nm, ϵ = 45 mM−1 cm−1 for compound I with mCPBA, and λmax = 408 nm, ϵ = 77 mM−1 cm−1 for compound II with mCPBA). The compound I-mCPBA intermediate (shown by near infrared spectroscopy to be identical to P965) decayed either to compound II in a catalytic cycle (k3 = 6.4 × 10−3 s−1) or, in a competing inactivation reaction, to verdohemoprotein (ki = 3.3 × 10−3 s−1). Thus, a partition ratio of r = 2 is obtained for the inactivation of ferric HRP-C by mCPBA. The intermediate formed from compound II with mCPBA is not part of the inactivation pathway and only decays via the catalytic cycle to give resting, ferric enzyme (k5 = 1.0 × 10−3 s−1). The data are compared with those from earlier steady-state kinetic studies and demonstrate the importance of single turnover experiments. The results are discussed in terms of the physiologically relevant reactions of plant peroxidases with hydrogen peroxide.