Reactivity of horseradish peroxidase compound II toward substrates: kinetic evidence for a two-step mechanism.
Transient kinetic analysis of biphasic, single turnover data for the reaction of 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with horseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M(-)(1) s(-)(1)) prior to rate-limiting electron transfer (k(+6) = 42.1 s(-)(1)). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to minimize absorbance changes in the Soret region due to the accumulation of ABTS cation radicals. A steady-state kinetic analysis of the reaction confirmed that the reduction of HRPC compound II by this substrate is rate-limiting in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorbate in the assay medium to minimize the effects of nonenzymic reactions involving phenol-derived radical products. This enabled the initial rates of o-diphenol oxidation at different hydrogen peroxide and o-diphenol concentrations to be determined from the lag period induced by the presence of ascorbate. The kinetic analysis resolved the reaction of HRPC compound II with o-diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphenols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s(-)(1)) is rate-limiting. The size and hydrophobicity of the o-diphenol substrates are correlated with their rate of binding to HRPC, while the electron density at the C-4 hydroxyl group predominantly influences the rate of electron transfer to the heme.
Rodríguez-López
J N
JN
Grupo de Investigación de Enzimología (GENZ), Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
Gilabert
M A
MA
Tudela
J
J
Thorneley
R N
RN
García-Cánovas
F
F
D001205:Ascorbic Acid / Q000737:chemistry; D052160:Benzothiazoles; D001665:Binding Sites; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry; D007202:Indicators and Reagents; D007700:Kinetics; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010636:Phenols / Q000737:chemistry; D019163:Reducing Agents / Q000737:chemistry; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000737:chemistry
D052160:Benzothiazoles; D007202:Indicators and Reagents; D010636:Phenols; D019163:Reducing Agents; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006861:Hydrogen Peroxide; D006735:Horseradish Peroxidase; D001205:Ascorbic Acid