Primary sequence and activity analyses of a catalase from Ascaris suum.
A complete cDNA encoding the catalase (EC 1.11.1.6) has been isolated from the parasitic nematode Ascaris suum (AsCAT). The active-site residues, the residues involved in ligand interaction, and NADPH-binding residues of the bovine liver catalase-type enzyme are highly conserved in the AsCAT predicted amino acid sequence. To confirm that the AsCAT cDNA encodes a functional enzyme, active recombinant protein (rAsCAT) was produced in a procaryotic expression system. The subunit molecular mass of the purified recombinant protein (rAsCAT) was determined to be approximately 60 kDa. According to gel filtration, the molecular mass of the active enzyme is 240 kDa, indicating that the catalase subunits form a homotetramer in solution. The optical spectrum of rAsCAT shows a typical ferric haem spectrum with a Soret band at 407 nm. Fluorescence spectroscopy demonstrates that rAsCAT binds NADPH. rAsCAT has catalase activity with hydrogen peroxide over a broad pH range, with a specific activity of 37,800 U mg-1. In addition to its catalase activity, rAsCAT displays peroxidase activity using the substrates t-butyl hydroperoxide and o-dianisidine. The haem ligands NaN3 and KCN caused a 50% inhibition of catalase activity at 9 and 19 microM, respectively. In the presence of a H2O2-generating system, catalase activity of rAsCAT was inhibited by 3-aminotriazole, phenolic compounds, and drugs.
Eckelt
V H
VH
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany. eckelt@hotmail.com
Liebau
E
E
Walter
R D
RD
Henkle-Dührsen
K
K
D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002374:Catalase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D018076:DNA, Complementary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009249:NADP; D006861:Hydrogen Peroxide; D002374:Catalase; D009195:Peroxidase