Kinetic study of the inactivation of ascorbate peroxidase by hydrogen peroxide.
The activity of ascorbate peroxidase (APX) has been studied with H(2)O(2) and various reducing substrates. The activity decreased in the order pyrogallol>ascorbate>guaiacol>2, 2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The inactivation of APX with H(2)O(2) as the sole substrate was studied. The number of H(2)O(2) 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 H(2)O(2) 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 microM) for H(2)O(2) was obtained in each case. However, the maximum rate constant (lambda(max)) obtained from the plots differed depending on the assay substrate. The presence of reducing substrate in addition to H(2)O(2) 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 H(2)O(2) 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.
Hiner
A N
AN
Departamento de Biología Vegetal (Fisiología Vegetal), Universidad de Murcia, E-30100 Espinardo, Murcia, Spain.
Rodríguez-López
J N
JN
Arnao
M B
MB
Lloyd Raven
E
E
García-Cánovas
F
F
Acosta
M
M
D060387:Ascorbate Peroxidases; D052160:Benzothiazoles; D002374:Catalase / Q000378:metabolism; D006139:Guaiacol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D008962:Models, Theoretical; D010084:Oxidation-Reduction; D018532:Pisum sativum / Q000201:enzymology; D010544:Peroxidases / Q000037:antagonists & inhibitors*; D011748:Pyrogallol / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D013379:Substrate Specificity; D013451:Sulfonic Acids / Q000494:pharmacology
D052160:Benzothiazoles; D011994:Recombinant Proteins; D013451:Sulfonic Acids; D011748:Pyrogallol; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006139:Guaiacol; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D002374:Catalase