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 t he cell. Recent studies have revealed that Plasmodium falciparum and h uman thioredoxin reductase represent a novel class of enzymes, which a re substantially different from the isofunctional prokaryotic Escheric hia coli enzyme. We identified the cysteines Cys(88) and Cys(93) as th e redox active disulfide and His(509) as the active site base \Gilberg er, T.-W., Walter, R.D. and Muller, S., J. Biol. Chem, 272 (1997) 2958 4-29589\, In addition to the active site thiols Cys(88) and Cys(93) th e P. falciparum enzyme has another pair of cysteines at the C-terminus : Cys(535) and Cys(540). To assess the possible role of these peripher al cysteines in the catalytic process the single mutants PfTrxRC535A a nd PfTrxRC540A, the double mutant PfTrxRC535AC540A and the deletion mu tant PfTrxR Delta 9 (C-terminal deletion of the last nine amino acids) were constructed. All mutants are defective in their thioredoxin redu ction activity, although they still show reactivity with 5,5'-dithiobi s (2-nitrobenzoate). These data imply that the C-terminal cysteines ar e crucially involved in substrate coordination and/or electron transfe r during reduction of the peptide substrate. (C) 1998 Federation of Eu ropean Biochemical Societies.