CATALASE ACTIVITY OF CHLOROPEROXIDASE AND ITS INTERACTION WITH PEROXIDASE-ACTIVITY

The catalatic activity of chloroperoxidase (CPO) was demonstrated to e xhibit saturation kinetics under steady-state conditions, which were n ot observed with catalase under comparable conditions. Results were ob tained using reaction mixtures of CPO and H2O2 at pH 6.2, rapid spectr al scan and single wavelength measurements, and transient- and steady- state reaction conditions. The observed rectangular hyperbolae (measur ement of rates of disappearance of H2O2 and appearance of O-2) could b e fit quantitatively to nu/[CPO](0)=B-1[H2O2]/B-2 + [H2O2] where nu is rate of O-2 evolution, [CPO](0) is total enzyme concentration, B-1 = (9 +/- 1) x 10(2) s(-1) and B-2 = (3.3 +/- 0.4) x 10(-3) M. The result s indicated formation of a complex of compound I (CPO-I) and H2O2, whi ch dissociated to native CPO, O-2, 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-phen ylenediamine (TMPD) under steady-state conditions. Attempts to determi ne Michaelis-Menten constants for the substrates TMPD and H2O2 gave ri se to apparently anomalous data. Our data showed that the modified pin g-pong mechanism established for horseradish peroxidase is applicable to the peroxidatic reaction catalyzed by chloroperoxidase. Both peroxi datic and catalatic reactions occurred in the reaction system containi ng H2O2, a reducing substrate, and CPO. A combined reaction mechanism was proposed for CPO-catalyzed reactions in which the modified ping-po ng mechanism was applicable for the peroxidatic reactions and the form ation of a CPO-I-H2O2 complex occurred for the catalatic reaction.