THE INACTIVATION AND CATALYTIC PATHWAYS OF HORSERADISH-PEROXIDASE WITH M-CHLOROPEROXYBENZOIC ACID - A SPECTROPHOTOMETRIC AND TRANSIENT KINETIC-STUDY

The kinetics of the catalytic cycle and irreversible inactivation of h orseradish peroxidase C (HRP-C) reacting with m-chloroperoxybenzoic ac id (mCPBA) have been studied by conventional and stopped-flow spectrop hotometry, mCPBA oxidized HRP-C to compound I with a second order-rate constant k(1) = 3.6 x 10(7) M(-1) s(-1) at pH 7.0, 25 degrees C. Exce ss mCPBA subsequently acted as a one-electron reducing substrate, conv erting compound I to compound II and compound II to resting, ferric en zyme. In both of these reactions, spectrally distinct, transient forms of the enzyme were observed (lambda(max) = 411 nm, epsilon = 45 mM(-1 ) cm(-1) for compound I with mCPB-4, and lambda(max) = 408 nm, epsilon = 77 mM(-1) cm(-1) for compound II with mCPBA), The compound I-mCPBA intermediate (shown by near infrared spectroscopy to be identical to P 965) decayed either to compound II in a catalytic cycle (k(3) = 6.4 x 10(-3) s(-1)) or, in a competing inactivation reaction, to verdohemopr otein (k(i) = 3.3 x 10(-3) s(-1)). Thus, a partition ratio of r = 2 is obtained for the inactivation of ferric HRP-C by mCPBA, The intermedi ate formed from compound II with mCPBA is not part of the inactivation pathway and only decays via the catalytic cycle to give resting, ferr ic enzyme (k(5) = 1.0 x 10(-3) s(-1)). The data are compared with thos e from earlier steady-state kinetic studies and demonstrate the import ance of single turnover experiments, The results are discussed in term s of the physiologically relevant reactions of plant peroxidases with hydrogen peroxide.