Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism.
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.
Gilabert
María Angeles
MA
GENZ: Grupo de Investigación de Enzimología, Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
Hiner
Alexander N P
AN
García-Ruiz
Pedro Antonio
PA
Tudela
José
J
García-Molina
Francisco
F
Acosta
Manuel
M
García-Cánovas
Francisco
F
Rodríguez-López
José Neptuno
JN
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
D000814:Aniline Compounds; D010636:Phenols; D019163:Reducing Agents; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D010100:Oxygen