RESUMO
Chloride ion (Cl-) effects on chloroperoxidase (CPO)-catalyzed peroxidation of catechol were used to probe the involvement of Cl- in CPO reactions. High concentrations of Cl- inhibit catechol peroxidation by competing with hydrogen peroxide (KI = 370 mM). However, at lower concentrations, Cl- is a linear competitive activator versus catechol (KDC = 35 mM). Addition of good halogenation substrates to the peroxidatic reaction mixture converts Cl- from a competitive activator to a competitive inhibitor. The KI (10 mM) for this halogenation substrate promoted Cl- inhibition is equivalent to the KM (11 mM) for Cl- in CPO-catalyzed halogenation reactions. During this inhibition, the halogenation substrate is consumed and, at the point where its consumption is complete, Cl- again becomes an activator. Also, at 2.0 mM hydrogen peroxide, CPOs chlorination reaction and its Cl- -activated peroxidatic reaction have similar apparent kcat values. All data are consistent with a mechanism in which Cl- competes with catechol for binding to CPO Compound I. Catechol binding initiates the Cl- -independent path, in which Compound I acts as the oxidizing agent for catechol. When Cl- binds to Compound I, it reacts to yield the enzymatic chlorinating intermediate which is responsible for either the oxidation of catechol in the Cl- -dependent path or the chlorination of substrates in the halogenation pathway. Cl- activation of the peroxidatic reaction is due to a shift from the Cl- -independent pathway to the Cl- -dependent process. The mechanism is unique in that exclusion of the substrate from its primary binding site leads to an increase in the catalytic efficiency of the reaction. This catechol-Cl- system also offers further potential for probing the specificity and chemistry of the key enzymatic intermediates in haloperoxidase-catalyzed reactions.