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 Cys88 and Cys93 as the redox active disulfide and His509 as the active site base [Gilberger, T.-W., Walter, R.D. and Müller, S., J. Biol. Chem. 272 (1997) 29584-29589]. In addition to the active site thiols Cys88 and Cys93 the P. falciparum enzyme has another pair of cysteines at the C-terminus: Cys535 and Cys540. To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant P/TrxRC535AC540A and the deletion mutant PfTrxRdelta9 (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.
Gilberger
T W
TW
Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Bergmann
B
B
Walter
R D
RD
Müller
S
S
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D007700:Kinetics; D016296:Mutagenesis / Q000235:genetics; D010084:Oxidation-Reduction; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013053:Spectrophotometry; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry* / Q000235:genetics
D004220:Disulfides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D013880:Thioredoxin-Disulfide Reductase; D003545:Cysteine