Catalase activity of chloroperoxidase and its interaction with peroxidase activity.
The catalatic activity of chloroperoxidase (CPO) was demonstrated to exhibit saturation kinetics under steady-state conditions, which were not observed with catalase under comparable conditions. Results were obtained using reaction mixtures of CPO and H2O2 at pH 6.2, rapid spectral scan and single wavelength measurements, and transient- and steady-state reaction conditions. The observed rectangular hyperbolae (measurement of rates of disappearance of H2O2 and appearance of O2) could be fit quantitatively to [equation: see text] where v is rate of O2 evolution, [CPO]0 is total enzyme concentration, B1 = (9 +/- 1) x 10(2) s-1, and B2 = (3.3 +/- 0.4) x 10(-3) M. The results indicated formation of a complex of compound I (CPO-I) and H2O2, which dissociated to native CPO, O2, and H2O with a rate constant of (9 +/- 1) x 10(2) s-1. The determination of the peroxidatic activity of CPO was performed using demethylation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) under steady-state conditions. Attempts to determine Michaelis-Menten constants for the substrates TMPD and H2O2 gave rise to apparently anomalous data. Our data showed that the modified ping-pong mechanism established for horseradish peroxidase is applicable to the peroxidatic reaction catalyzed by chloroperoxidase. Both peroxidatic and catalatic reactions occurred in the reaction system containing H2O2, a reducing substrate, and CPO. A combined reaction mechanism was proposed for CPO-catalyzed reactions in which the modified ping-pong mechanism was applicable for the peroxidatic reactions and the formation of a CPO-I-H2O2 complex occurred for the catalatic reaction.
Sun
W
W
Department of Chemistry, University of Alberta, Edmonton, Canada.
Kadima
T A
TA
Pickard
M A
MA
Dunford
H B
HB
D002374:Catalase / Q000378:metabolism*; D002384:Catalysis; D002711:Chloride Peroxidase / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D009195:Peroxidase / Q000378:metabolism*; D013772:Tetramethylphenylenediamine / Q000378:metabolism
D005609:Free Radicals; D006861:Hydrogen Peroxide; D002711:Chloride Peroxidase; D002374:Catalase; D009195:Peroxidase; D013772:Tetramethylphenylenediamine