Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme.
A hydroperoxidase purified from the halophilic archaeon Halobacterium halobium exhibited both catalase and peroxidase activities, which were greatly diminished in a low-salt environment. Therefore, the purification was carried out in 2 M NaCl. Purified protein exhibited catalase activity over the narrow pH range of 6.0 to 7.5 and exhibited peroxidase activity between pH 6.5 and 8.0. Peroxidase activity was maximal at NaCl concentrations above 1 M, although catalase activity required 2 M NaCl for optimal function. Catalase activity was greatest at 50 degrees C; at 90 degrees C, the enzymatic activity was 20% greater than at 25 degrees C. Peroxidase activity decreased rapidly above its maximum at 40 degrees C. An activation energy of 2.5 kcal (ca. 10 kJ)/mol was calculated for catalase, and an activation energy of 4.0 kcal (ca. 17 kJ)/mol was calculated for peroxidase. Catalase activity was not inhibited by 3-amino-1,2,4-triazole but was inhibited by KCN and NaN3 (apparent Ki [KiApp] of 50 and 67.5 microM, respectively). Peroxidative activity was inhibited equally by KCN and NaN3 (KiApp for both, approximately 30 microM). The absorption spectrum showed a Soret peak at 404 nm, and there was no apparent reduction by dithionite. A heme content of 1.43 per tetramer was determined. The protein has a pI of 3.8 and an M(r) of 240,000 and consists of four subunits of 60,300 each.
Brown-Peterson
N J
NJ
Department of Biochemistry and Molecular Biology, Mississippi State University 39762.
Salin
M L
ML
D002374:Catalase / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D019617:Halobacterium salinarum / Q000201:enzymology*; D006358:Hot Temperature; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D010544:Peroxidases / Q000302:isolation & purification / Q000378:metabolism*; D012492:Salts; D013053:Spectrophotometry
D012492:Salts; D006861:Hydrogen Peroxide; D010544:Peroxidases; D002374:Catalase