Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme.
A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min-1 (mg protein)-1. The dismutase obeyed Michaelis-Menten kinetics. The Vmax and Km calculated for chlorite were 2,200 U (mg protein)-1 and 170 microM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30 degrees C.
van Ginkel
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Akzo Nobel Central Research, Analytical and Environmental Chemistry Department, Velperweg 76, 6800 SB Arnhem, The Netherlands. Kees.C.G.vanginkel@Akzo.NL
Rikken
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Kroon
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Kengen
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D000640:Amitrole / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D002457:Cell Extracts / Q000037:antagonists & inhibitors; D002712:Chlorides / Q000378:metabolism*; D003486:Cyanides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism
D001386:Azides; D002457:Cell Extracts; D002712:Chlorides; D003486:Cyanides; D006898:Hydroxylamines; D019811:Hydroxylamine; D010088:Oxidoreductases; D010100:Oxygen; C001599:chlorite; D000640:Amitrole