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Examining the role of glutamic acid 183 in chloroperoxidase catalysis

Author: Yi, X. · Conesa, A. · Punt, P.J. · Hager, L.P.
Source:Journal of Biological Chemistry, 16, 278, 13855-13859
Identifier: 237045
doi: doi:10.1074/jbc.M210906200
Keywords: Biology · Biotechnology · Catalysis · Genetic engineering · Organic acids · X ray crystallography · Active sites · Enzymes · Chloride peroxidase · Clutamic acid · Histidine · Hydrogen peroxide · Natural product · Epoxidase · Oxidoreductase · Amino acid substitution · Animal cell · Asymmetric catalysis · Chlorination · Enzyme active site · Enzyme activity · Epoxidation · Genetic analysis · Hydrophobicity · Nonhuman · Plasmid · Site directed mutagenesis · X ray crystallography · Chemistry · Circular dichroism · Enzymology · Immunoblotting · Ion exchange chromatography · Isoelectric focusing · Metabolism · PH · polyacrylamide gel electrophoresis · Animalia · Aspergillus · Aspergillus niger · Catalase · Catalysis · Chloride Peroxidase · Chlorine · Chromatography, Ion Exchange · Circular Dichroism · Crystallography, X-Ray · Electrophoresis, Polyacrylamide Gel · Fungi · Glutamic Acid · Histidine · Hydrogen-Ion Concentration · Immunoblotting · Isoelectric Focusing · Mutation · Oxidoreductases · Plasmids


Site-directed mutagenesis has been used to investigate the role of glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray crystallographic structure of chloroperoxidase, Glu-183 is postulated to function on distal side of the heme prosthetic group as an acid-base catalyst in facilitating the reaction between the peroxidase and hydrogen peroxide with the formation of Compound I. In contrast, the other members of the heme peroxidase family use a histidine residue in this role. Plasmids have now been constructed in which the codon for Glu-183 is replaced with a histidine codon. The mutant recombinant gene has been expressed in Aspergillus niger. An analysis of the produced mutant gene shows that the substitution of Glu-183 with a His residue is detrimental to the chlorination and dismutation activity of chloroperoxidase. The activity is reduced by 85 and 50% of wild type activity, respectively. However, quite unexpectedly, the epoxidation activity of the mutant enzyme is significantly enhanced ∼2.5-fold. These results show that Glu-183 is important but not essential for the chlorination activity of chloroperoxidase. It is possible that the increased epoxidation of the mutant enzyme is based on an increase in the hydrophobicity of the active site.