Steady-state kinetics of thiocyanate oxidation catalyzed by human salivary peroxidase

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Abstract
A steady-state kinetic analysis was made of thiocyanate (SCN-) oxidation catalyzed by human peroxidase (SPO) isolated from parotid saliva. For comparative purposes, bovine lactoperoxidase (LPO) was also studied. Both enzymes followed the classical Theorell-Chance mechanism under the initial conditions [H2O2] < 0.2mM, [SCN-] < 10 mM, and pH < 6.0. The pH-independent rate constants (k1) for the formation of compound I were estimated to be 8 .times. 106 M-1 s-1 (SD =1, n = 18) for LPO and 5 .times. 106 M-1 s-1 (SD = 1, n = 11) for SPO. The pH-independent second-order rate constants (k4) for the oxidation of thiocyanate by compound I were estimated to be 5 .times. 106 M-1 s-1 (SD = 1, n = 18) for LPO and 9 .times. 106 M-1 s-1 (SD = 2, n = 11) for SPO. Both enzymes were inhibited by SCN- at pH < 6. The pH-independent equilibrium constant (Ki) for the formation of the inhibited enzyme-SCN- complex was estimated to be 24 M-1 (SD = 12, n = 8) for LPO and 44 M-1 (SD = 4, n = 10) for SPO. An apparent pH dependence of the estimated values for k4 and Ki for both LPO and SPO was consistent with a mechanism based on assumptions that protonation of compound I was necessary for the SCN- peroxidation step, that a second protonation of compound I gave an inactive form, and that the inhibited enzyme-SCN- complex could be further protonated to give another inactive form. The pKa for the formation of this latter complex was estimated to be 6.0 for both enzymes, which corresponds to the pKa of a histidine imidazole group. Maximum SCN- peroxidation rates were observed for SPO in the pH range 5-6.

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