Examining the role of glutamic acid 183 in chloroperoxidase catalysis.
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 approximately 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.
Yi
Xianwen
X
Department of Biochemistry, University of Illinois at Urbana-Champaign, 61801, USA.
Conesa
Ana
A
Punt
Peter J
PJ
Hager
Lowell P
LP
D001234:Aspergillus niger / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002384:Catalysis; D002711:Chloride Peroxidase / Q000737:chemistry* / Q000378:metabolism*; D002713:Chlorine / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D005658:Fungi / Q000201:enzymology; D018698:Glutamic Acid / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007525:Isoelectric Focusing; D009154:Mutation; D010088:Oxidoreductases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism
D018698:Glutamic Acid; D006639:Histidine; D002713:Chlorine; D010088:Oxidoreductases; C023111:epoxidase; D002711:Chloride Peroxidase; D002374:Catalase