The chloride-activated peroxidation of catechol as a mechanistic probe of chloroperoxidase reactions. Competitive activation as evidence for a catalytic chloride binding site on compound I.
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.
Libby
R D
RD
Chemistry Department, Colby College, Waterville, Maine 04901.
Rotberg
N S
NS
Emerson
J T
JT
White
T C
TC
Yen
G M
GM
Friedman
S H
SH
Sun
N S
NS
Goldowski
R
R
D002396:Catechols / Q000378:metabolism*; D002711:Chloride Peroxidase / Q000378:metabolism*; D002712:Chlorides / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010544:Peroxidases / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry
D002396:Catechols; D002712:Chlorides; D006861:Hydrogen Peroxide; D010544:Peroxidases; D002711:Chloride Peroxidase; C034221:catechol