Permutation of the Active Site Motif of Tryparedoxin 2
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Peter Steinert
Abstract
Tryparedoxins (TXN) are thioredoxinrelated proteins which, as trypanothione:peroxiredoxin oxidoreductases, constitute the trypanothionedependent antioxidant defense and may also serve as substrates for ribonucleotide reductase in trypanosomatids. The active site motif of TXN2, [40]WCPPCR[45], of Crithidia fasciculata was mutated by sitedirected mutagenesis and eight corresponding muteins were expressed in E. coli as terminally Histagged proteins, purified to homogeneity by nickel chelate chromatography, and characterized in terms of specific activity, specificity and, if possible, kinetics. Exchange of Cys41 and Cys44 by serine yielded inactive products confirming their presumed involvement in catalysis. Exchange of Arg45 by aspartate resulted in loss of activity, suggesting an activation of active site cysteines by the positive charge of Arg45. Substitution of Trp40 by phenylalanine or tyrosine resulted in moderate decrease of specific activity, as did exchange of Pro42 by glycine. Kinetic analysis of these three muteins revealed that primarily the reaction with trypanothione is affected by the mutations. Simulation of thioredoxin or glutaredoxin like active sites in TXN2 (P42G and W40T/P43Y, respectively) did not result in thioredoxin or glutaredoxin like activities. These data underscore that TXNs, although belonging to the thioredoxin superfamily, represent a group of enzymes distinct from thioredoxins and glutaredoxins in terms of specificity, and appear attractive as molecular targets for the design of trypanocidal compounds.
Copyright © 2000 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- Interaction of E. coli Single-Stranded DNA Binding Protein (SSB) with Exonuclease I. The Carboxy-Terminus of SSB Is the Recognition Site for the Nuclease
- p38/SAPK2-Dependent Gene Expression in Jurkat T Cells
- The First Laminin G-Type Domain in the SHBG-Like Region of Protein S Contains Residues Essential for Activation of the Receptor Tyrosine Kinase Sky
- Permutation of the Active Site Motif of Tryparedoxin 2
- Structural Investigations of the Highly Flexible Recombinant Ribosomal Protein L12 from Thermotoga maritima
- Polyhistidine-Tagged Hepatitis B Core Particles as Carriers of HIV-1/gp120 Epitopes of Different HIV-1 Subtypes
- In Vitro Evolution of Ligands for HCV-Specific Serum Antibodies
- Stability of Bacteriophage T4 Short Tail Fiber
- Analysis of the RNase T1 Mediated Cleavage of an Immobilized Gapped Heteroduplex via Fluorescence Correlation Spectroscopy
- Binding of Selenoprotein P to Heparin: Characterization with Surface Plasmon Resonance
- Biotin-Avidin Microplate Assay for the Quantitative Analysis of Enzymatic Methylation of DNA by DNA Methyltransferases
