Identification of Acid-Base Catalytic Residues of High-Mr Thioredoxin Reductase from Plasmodium falciparum

Paul J. McMillan,Katja Becker,L. David Arscott,Charles H. Williams,David P. Ballou,Sylke Müller

doi:10.1074/jbc.m601141200

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.

Highlights

Ribonucleotide reductase, methionine oxide reductase, 2-Cys peroxiredoxins, and a number of transcription factors [1,2,3,4]
High-Mr Thioredoxin reductase (TrxR) is present in mammals, insects, worms, and the malaria parasite, Plasmodium falciparum [5, 6, 15, 19, 20]
This study focuses on acid-base catalysis in the reaction cycle of the high-Mr TrxR from the malaria parasite, P. falciparum

Summary

Introduction

Ribonucleotide reductase, methionine oxide reductase, 2-Cys peroxiredoxins, and a number of transcription factors [1,2,3,4]. Because of increasing resistances of the parasites against antimalarial drugs, new and better chemotherapies are urgently required [9, 10]. Plasmodium lacks both glutathione peroxidase and catalase, indicating that the thioredoxin system is important for multiple roles, including reduction of glutathione, protection against oxidative stress, and biosynthesis of thymine. To date three compounds showing antimalarial activity as well as specific inhibition of PfTrxR over human TrxR are available [13, 14] To facilitate these investigations, a colorimetric microtiter assay has been developed that is suitable for high throughput inhibitor screening on PfTrxR [14]. The reduction of Eox by NADPH is a priming reaction

Results

Discussion

Conclusion