Primary sequence and activity analyses of a catalase from Ascaris suum

Abstract

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 ∼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 NaN 3 and KCN caused a 50% inhibition of catalase activity at 9 and 19 μM, respectively. In the presence of a H 2O 2-generating system, catalase activity of rAsCAT was inhibited by 3-aminotriazole, phenolic compounds, and drugs.