title: Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism.
abstract: The catalytic constant (k(cat)) and the second-order association constant of compound II with reducing substrate (k(5)) of horseradish peroxidase C (HRPC) acting on phenols and anilines have been determined from studies of the steady-state reaction velocities (V(0) vs. [S(0)]). Since k(cat)=k(2)k(6)/k(2)+k(6), and k(2) (the first-order rate constant for heterolytic cleavage of the oxygen-oxygen bond of hydrogen peroxide during compound I formation) is known, it has been possible to calculate the first-order rate constant for the transformation of each phenol or aniline by HRPC compound II (k(6)). The values of k(6) are quantitatively correlated to the sigma values (Hammett equation) and can be rationalized by an aromatic substrate oxidation mechanism in which the substrate donates an electron to the oxyferryl group in HRPC compound II, accompanied by two proton additions to the ferryl oxygen atom, one from the substrate and the other the protein or solvent. k(6) is also quantitatively correlated to the experimentally determined (13)C-NMR chemical shifts (delta(1)) and the calculated ionization potentials, E (HOMO), of the substrates. Similar dependencies were observed for k(cat) and k(5). From the kinetic analysis, the absolute values of the Michaelis constants for hydrogen peroxide and the reducing substrates (K(M)(H(2)O(2)) and K(M)(S)), respectively, were obtained.
authors.affiliation: GENZ: Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
authors.forename: María Angeles
authors.initials: MA
authors.lastname: Gilabert
authors.forename: Alexander N P
authors.initials: AN
authors.lastname: Hiner
authors.forename: Pedro Antonio
authors.initials: PA
authors.lastname: García-Ruiz
authors.forename: José
authors.initials: J
authors.lastname: Tudela
authors.forename: Francisco
authors.initials: F
authors.lastname: García-Molina
authors.forename: Manuel
authors.initials: M
authors.lastname: Acosta
authors.forename: Francisco
authors.initials: F
authors.lastname: García-Cánovas
authors.forename: José Neptuno
authors.initials: JN
authors.lastname: Rodríguez-López
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010636:Phenols / Q000378:metabolism*; D019163:Reducing Agents / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D000814:Aniline Compounds; D010636:Phenols; D019163:Reducing Agents; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010100:Oxygen