Study of ceruloplasmin oxidase activity. The effect of pH

To find out the mechanism of ceruloplasmin (CP) oxidase activity CP interaction with organic substrates adrenaline (AD), catechol, p-phenylenediamine) and Fe2+ was investigated. CP was shown to interact with the above substrates according to the Theorell-Chance mechanism to form a kinetically insignificant ternary complex. The oxygen molecule binds first to CP followed by the molecule of electron donor: the inhibition of enzymatic oxidation by the reaction product is competitive. The effects of pH on kinetic parameters (Ksm, Vs) of oxidation reactions of AD and Fe2+ in the presence of CP were examined. AD was shown to be able to bind both to the protonated and to the non-protonated form of CP whereas Fe2+ interacts only with the protonated form: the rate of catalytic event (Vs) is influenced by pH in the presence of AD whereas in the presence of Fe2+ only binding to CP (Ksm) is affected by pH. Kinetic schemes describing the order of binding of hydrogen ions in the course of above reactions are proposed.