Studies on the Mechanism of the Chlorination Reactions Catalyzed by Chloroperoxidase and by Horseradish Peroxidase With Chlorite
Shahangian, Shahram
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https://hdl.handle.net/2142/70509
Description
Title
Studies on the Mechanism of the Chlorination Reactions Catalyzed by Chloroperoxidase and by Horseradish Peroxidase With Chlorite
Author(s)
Shahangian, Shahram
Issue Date
1982
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Abstract
Chloroperoxidase catalyzes the dismutation of chlorite-forming chloride, chlorine dioxide, chlorate, and oxygen as products. Chloroperoxidase also catalyzes the decomposition of chlorine dioxide. Chloride, chlorate, and oxygen are the products of the decomposition of chlorine dioxide. The optimum pH for the enzymic decomposition of both chlorite and chlorine dioxide is approximately 2.75. At this pH, 1 mole of chlorine dioxide is dismutated to 0.3 mole of chloride, 0.7 mole of chlorate, and 0.17 mole of oxygen. At the same pH, the complete decomposition of 1 mole of chlorite yields 0.4 mole of chloride, 0.6 mole of chlorate, and 0.13 mole of oxygen. Kinetic parameters for the chlorite reaction have been determined. The K(,m) value for chlorite obtained from various kinetic plots was about 10 mM. The catalytic rate constant for the formation of chlorine dioxide from chlorite was about 70,000 s('-1).
Chloroperoxidase and horseradish peroxidase catalyze the chlorination of 4,4-dimethyl-2-chloro-1,3-cyclohexanedione (monochlorodimedone) to form 4,4-dimethyl-2,2-dichloro-1,3-cyclohexanedione (dichlorodimedone) using chlorite. The kinetics of both reactions have been studied yielding quite similar kinetic parameters for the two enzymes. The catalytic rate constant was about 2,400 s('-1) for chloroperoxidase and approximately 1,000 s('-1) for horseradish peroxidase.
The kinetics of the chemical reaction of monochlorodimedone with chlorite was directly compared with the kinetics of the enzymic reactions of monochlorodimedone with chlorite. At micromolar enzyme concentrations the enzymic reactions were faster by more than two orders of magnitude. These observations, substrate specificity studies using methionine and thiourea as co-substrates, and products analyses eliminate the involvement of chlorine dioxide in the enzymic reactions.
Horseradish peroxidase reacts with sodium {('36)Cl}chlorite at pH 10.7 to form a ('36)Cl-labeled horseradish peroxidase. This intermediate can be separated from small molecules by chromarography on a Sephadex G-10 column. After fractionation, 65 to 93% of ('36)Cl in the reaction mixture remains associated with the enzyme. The ('36)Cl-labeled enzyme reacts with monochlorodimedone at pH 4 to transfer ('36)Cl from the enzyme to the halogen acceptor molecule. A chlorine oxide ligand on a ferryl heme iron protoporphyrin IX is proposed for the structure of this intermediate, referred to as Compound X.
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