Kinetics of Spanish broom peroxidase obeys a Ping-Pong Bi–Bi mechanism with competitive inhibition by substrates

In plants, adverse conditions often induce an increase in reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). H2O2 is reduced to water, and thus becomes detoxified by enzymes such as Cytisus multiflorus peroxidase (CMP). Here, the steady-state kinetics of the H2O2-supported oxidation of different organic substrates by CMP was investigated. Analysis of the initial rates vs. H2O2 and reducing substrate concentrations proved to be consistent with a substrate-inhibited Ping-Pong Bi–Bi reaction mechanism. The phenomenological approach 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 KmH2O2, KmAH2, kcat, KSIH2O, KSIAH2 and of the microscopic rate constants, k1 and k3, of the shared three-step catalytic cycle of peroxidases.