title: Identification of acid-base catalytic residues of high-Mr thioredoxin reductase from Plasmodium falciparum.
abstract: High-M(r) thioredoxin reductase from the malaria parasite Plasmodium falciparum (PfTrxR) contains three redox active centers (FAD, Cys-88/Cys-93, and Cys-535/Cys-540) that are in redox communication. The catalytic mechanism of PfTrxR, which involves dithiol-disulfide interchanges requiring acid-base catalysis, was studied by steady-state kinetics, spectral analyses of anaerobic static titrations, and rapid kinetics analysis of wild-type enzyme and variants involving the His-509-Glu-514 dyad as the presumed acid-base catalyst. The dyad is conserved in all members of the enzyme family. Substitution of His-509 with glutamine and Glu-514 with alanine led to TrxR with only 0.5 and 7% of wild type activity, respectively, thus demonstrating the crucial roles of these residues for enzymatic activity. The H509Q variant had rate constants in both the reductive and oxidative half-reactions that were dramatically less than those of wild-type enzyme, and no thiolateflavin charge-transfer complex was observed. Glu-514 was shown to be involved in dithiol-disulfide interchange between the Cys-88/Cys-93 and Cys-535/Cys-540 pairs. In addition, Glu-514 appears to greatly enhance the role of His-509 in acid-base catalysis. It can be concluded that the His-509-Glu-514 dyad, in analogy to those in related oxidoreductases, acts as the acid-base catalyst in PfTrxR.
authors.affiliation: Institute of Biomedical and Life Sciences, Infection and Immunity, Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow G12 8TA, Scotland, UK.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: McMillan
authors.forename: L David
authors.initials: LD
authors.lastname: Arscott
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.forename: Katja
authors.initials: K
authors.lastname: Becker
authors.forename: Charles H
authors.initials: CH
authors.lastname: Williams
authors.forename: Sylke
authors.initials: S
authors.lastname: Müller
mesh_terms: D000143:Acids; D000468:Alkalies; D000818:Animals; D002384:Catalysis; D004227:Dithionite; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D013057:Spectrum Analysis; D013880:Thioredoxin-Disulfide Reductase / Q000235:genetics / Q000378:metabolism*; D017186:Titrimetry
chemical_list: D000143:Acids; D000468:Alkalies; D004227:Dithionite; D009249:NADP; D013880:Thioredoxin-Disulfide Reductase