The role of the C-terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum

The thioredoxin system is one of the major thiol reducing systems of the cell. Recent studies have revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, which are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. We identified the cysteines Cys(88) and Cys(93) as the redox active disulfide and His(509) as the active site base \Gilberger, T.-W., Walter, R.D. and Muller, S., J. Biol. Chem, 272 (1997) 29584-29589\, In addition to the active site thiols Cys(88) and Cys(93) the P. falciparum enzyme has another pair of cysteines at the C-terminus: Cys(535) and Cys(540). To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant PfTrxRC535AC540A and the deletion mutant PfTrxR Delta 9 (C-terminal deletion of the last nine amino acids) were constructed. All mutants are defective in their thioredoxin reduction activity, although they still show reactivity with 5,5'-dithiobis (2-nitrobenzoate). These data imply that the C-terminal cysteines are crucially involved in substrate coordination and/or electron transfer during reduction of the peptide substrate. (C) 1998 Federation of European Biochemical Societies.