The activity of ascorbate peroxidase (APX) has been studied with H2O2 and various reducing substrates. The activity decreased in the order pyrogallol > ascorbate > guaiacol > 2,2ʹ-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The inactivation of APX with H2O2 as the sole substrate was studied. The number of H2O2 molecules required for maximal inactivation of the enzyme was determined as approx. 2.5. Enzymic activity of approx. 20% of the original remained at the end of the inactivation process (i.e. approx. 20% resistance) when ascorbate or ABTS was used as the substrate in activity assays. With pyrogallol or guaiacol no resistance was seen. Inactivation by H2O2 followed over time with ascorbate or pyrogallol assays exhibited single-exponential decreases in enzymic activity. Hyperbolic saturation kinetics were observed in both assay systems; a similar dissociation constant (0.8 μM) for H2O2 was obtained in each case. However, the maximum rate constant (λmax) obtained from the plots differed depending on the assay substrate. The presence of reducing substrate in addition to H2O2 partly or completely protected the enzyme from inactivation, depending on how many molar equivalents of reducing substrate were added. An oxygen electrode system has been used to confirm that APX does not exhibit a catalase-like oxygen-releasing reaction. A kinetic model was developed to interpret the experimental results; both the results and the model are compared and contrasted with previously obtained results for horseradish peroxidase C. The kinetic model has led us to the conclusion that the inactivation of APX by H2O2 represents an unusual situation in which no enzyme turnover occurs but there is a partition of the enzyme between two forms, one inactive and the other with activity towards reducing substrates such as ascorbate and ABTS only. The partition ratio is less than 1.
Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide
- Views Icon Views
- Share Icon Share
Alexander N. P. HINER, José Neptuno RODRÍGUEZ-LÓPEZ, Marino B. ARNAO, Emma LLOYD RAVEN, Francisco GARCÍA-CÁNOVAS, Manuel ACOSTA; Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide. Biochem J 1 June 2000; 348 (2): 321–328. doi: https://doi.org/10.1042/bj3480321
Download citation file: