title: The inactivation of horseradish peroxidase isoenzyme A2 by hydrogen peroxide: an example of partial resistance due to the formation of a stable enzyme intermediate.
abstract: The inactivation of horseradish peroxidase A2 (HRP-A2) with H2O2 as the sole substrate has been studied. In incubation experiments it was found that the fall in HRP-A2 activity was non-linearly dependent on H2O2 concentrations and that a maximum level of inactivation of approximately 80% (i.e. approximately 20% residual activity) was obtained with 2,000 or more equivalents of H2O2. Further inactivation was only induced at much higher H2O2 concentrations. Spectral changes during incubations of up to 5 days showed the presence of a compound III-like species whose abundance was correlated to the level of resistance observed. Inactivation was pH dependent, the enzyme being much more sensitive under acid conditions. A partition ratio (r1 approximately equals 1,140 at pH 6.5) between inactivation and catalysis was calculated from the data. The kinetics of inactivation followed single exponential time curves and were H2O2 concentration dependent. The apparent maximum rate constant of inactivation was lambdamax=3.56+/-0.07x10(-4)s(-1) and the H2O2 concentration required to give lambdamax/2 was K2=9.94+/-0.52 mM. The relationship lambdamax<ki has been shown to apply and thus the rate constant of inactivation has been calculated as ki=1.9x10(-3)s(-1). HRP-A2 possessed catalase-like oxygen gas-releasing activity, the catalytic constant being k3=2.2 s(-1), and the affinity for H2O2 as K2=23 mM. Catalase-like activity was pH dependent and favoured under more basic conditions. A mechanistic model has been developed and used to explain the behaviour of HRP-A2. The model suggests that, in common with HRP-C, mechanism-based (suicide) inactivation is being observed but that a fraction of the HRP-A2 is protected from inactivation in the form of a modified compound III species.
authors.affiliation: Departamento de Biología Vegetal (Fisiología Vegetal), Universidad de Murcia, Espinardo, Spain.
authors.forename: A N
authors.initials: AN
authors.lastname: Hiner
authors.forename: J
authors.initials: J
authors.lastname: Hernández-Ruiz
authors.forename: J N
authors.initials: JN
authors.lastname: Rodríguez-López
authors.forename: M B
authors.initials: MB
authors.lastname: Arnao
authors.forename: R
authors.initials: R
authors.lastname: Varón
authors.forename: F
authors.initials: F
authors.lastname: García-Cánovas
authors.forename: M
authors.initials: M
authors.lastname: Acosta
mesh_terms: D002374:Catalase / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016877:Oxidants / Q000737:chemistry; D018384:Oxidative Stress; D013774:Tetranitromethane / Q000737:chemistry; D013997:Time Factors
chemical_list: D007527:Isoenzymes; D016877:Oxidants; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D002374:Catalase; D013774:Tetranitromethane