Krnajski Z, Gilberger T W, Walter R D, Müller S
Bernhard Nocht Institute for Tropical Medicine, Biochemical Parasitology, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany.
Mol Biochem Parasitol. 2001 Feb;112(2):219-28. doi: 10.1016/s0166-6851(00)00372-8.
The thioredoxin system consists of the NADPH dependent disulphide oxidoreductase thioredoxin reductase (TrxR) which catalyses the reduction of the small protein thioredoxin. This system is involved in a variety of biological reactions including the reduction of deoxyribonucleotides, transcription factors and hydrogen peroxide. In recent years the TrxR of the malaria parasite Plasmodium falciparum was isolated and characterised using model substrates like 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and Escherichia coli thioredoxin. Here we report on the isolation of a cDNA encoding for P. falciparum thioredoxin (PfTrx) and the expression and characterisation of the recombinant protein, the natural substrate of PfTrxR. The deduced amino acid sequence of PfTrx encodes for a polypeptide of 11715 Da and possesses the typical thioredoxin active site motif CysGlyProCys. Both cysteine residues are essential for catalytic activity of the protein, as shown by mutational analyses. Steady state kinetic analyses with PfTrxR and PfTrx in several coupled assay systems resulted in K(m)-values for PfTrx in the range of 0.8--2.1 microM which is about 250-fold lower than for the model substrate E. coli thioredoxin. Since the turnover of both substrates is similar, the catalytic efficiency of PfTrxR to reduce the isolated PfTrx is at least 250-fold higher than to reduce E. coli thioredoxin. PfTrx contains a cysteine residue in position 43 in addition to the active-site cysteine residues, which is partially responsible for dimer formation of the protein as demonstrated by changing this amino acid into an alanine residue. Using DTNB we showed that all three cysteine residues present in PfTrx are accessible to modification by this compound. Surprisingly the first cysteine residue of the active site motif (Cys30) is less accessible than the second cysteine (Cys33), which is highly prone to the modification. These results suggest a difference in the structure and reaction mechanism of PfTrx compared to other known thioredoxins.
硫氧还蛋白系统由依赖烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的二硫化物氧化还原酶硫氧还蛋白还原酶（TrxR）组成，该酶催化小蛋白硫氧还蛋白的还原反应。该系统参与多种生物反应，包括脱氧核糖核苷酸、转录因子和过氧化氢的还原反应。近年来，利用5,5'-二硫代双（2-硝基苯甲酸）（DTNB）和大肠杆菌硫氧还蛋白等模型底物，分离并鉴定了疟原虫恶性疟原虫的TrxR。在此，我们报告了编码恶性疟原虫硫氧还蛋白（PfTrx）的cDNA的分离，以及PfTrxR的天然底物重组蛋白的表达和鉴定。PfTrx推导的氨基酸序列编码一个11715 Da的多肽，并具有典型的硫氧还蛋白活性位点基序CysGlyProCys。如突变分析所示，两个半胱氨酸残基对该蛋白的催化活性至关重要。在几个偶联分析系统中对PfTrxR和PfTrx进行稳态动力学分析，结果显示PfTrx的米氏常数（K(m)）值在0.8 - 2.1 microM范围内，这比模型底物大肠杆菌硫氧还蛋白的K(m)值低约250倍。由于两种底物的周转相似，PfTrxR还原分离出的PfTrx的催化效率比还原大肠杆菌硫氧还蛋白至少高250倍。除了活性位点半胱氨酸残基外，PfTrx在第43位还含有一个半胱氨酸残基，将该氨基酸突变为丙氨酸残基的实验表明，该残基部分负责该蛋白的二聚体形成。使用DTNB我们发现，PfTrx中存在的所有三个半胱氨酸残基都可被该化合物修饰。令人惊讶的是，活性位点基序的第一个半胱氨酸残基（Cys30）比第二个半胱氨酸（Cys33）更不易被修饰，而第二个半胱氨酸极易被修饰。这些结果表明，与其他已知硫氧还蛋白相比，PfTrx在结构和反应机制上存在差异。
