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 resi dues, the residues involved in ligand interaction, and NADPH-binding r esidues 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 b e similar to 60 kDa. According to gel filtration, the molecular mass o f 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. Fluorescen ce spectroscopy demonstrates that rAsCAT binds NADPH. rAsCAT has catal ase activity with hydrogen peroxide over a broad pH range, with a spec ific activity of 37 800 U mg(-1). In addition to its catalase activity , rAsCAT displays peroxidase activity using the substrates t-butyl hyd roperoxide and o-dianisidine. The haem ligands NaN3 and KCN caused a 5 0% inhibition of catalase activity at 9 and 19 mu M, respectively. In the presence of a H2O2-generating system, catalase activity of rAsCAT was inhibited by 3-aminotriazole, phenolic compounds, and drugs. (C) 1 998 Elsevier Science B.V. All rights reserved.