TRYPANOTHIONE REDUCTASE FROM TRYPANOSOMA-CRUZI - PURIFICATION AND CHARACTERIZATION OF THE CRYSTALLINE ENZYME
TRYPANOTHIONE REDUCTASE FROM TRYPANOSOMA-CRUZI - PURIFICATION AND CHARACTERIZATION OF THE CRYSTALLINE ENZYME
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DOI：
10.1111/j.1432-1033.1987.tb11002.x
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发表时间：
1987-04-01
期刊：
影响因子：
--
通讯作者：
SCHIRMER, RH
中科院分区：
文献类型：
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作者：
KRAUTHSIEGEL, RL;ENDERS, B;SCHIRMER, RH
克氏锥虫的锥虫硫酮还原酶和人谷胱甘肽还原酶（一种已知三维结构的酶）之间的结构差异为针对恰加斯病的合理药物设计提供了机会。作为寄生虫酶分析的第一步，我们报告其纯化和表征。1. 2.2从33克湿重的培养的外鞭毛体或从4克冻干细胞中提取10毫克锥虫硫酮还原酶。黄酶纯化2400倍，在三个步骤的总收率为45%的同质。2.该酶是一种二聚体，亚基Mr为50，000。使用NADPH（Km=5 μ M）和二硫化物锥虫硫酮（Km=45 μ M）作为底物，估计周转数为14200 min-1。锥虫硫酮还原酶（寄生酶）和谷胱甘肽还原酶（宿主酶）对各自的二硫化物底物表现出相互排斥的特异性。3.当筛选细胞培养物或柱洗脱液中是否存在锥虫硫酮还原酶时，使用基于Ellman试剂作为指示剂的微量测定法。从大肠杆菌中分离的区域异构体谷胱甘肽亚精胺二硫化物的混合物作为本微量测定的底物。4.在实验上，酶的催化循环可细分为半反应Eox + ADH + H+ →。EN 2 + NADP+和EH 2+二硫化物锥虫硫酮→EOX +二氢锥虫硫酮。在2 M（NH 4）2SO 4存在下，结晶酶也是如此。5.氧化形式（Eox）和双电子还原形式（EH 2）的锥虫硫酮还原酶的光谱性质与人谷胱甘肽还原酶的光谱性质非常相似。这两种蛋白质在催化位点都含有黄素和氧化还原活性的亚硫酸盐。在Eox还原为EH 2后，锥虫酶还原酶可以通过特异性烷基化新生活性位点硫醇之一而失活。
The structural differences between trypanothione reductase of Trypanosoma cruzi and human glutathione reductase, an enzyme of known three-dimensional structure, offer an opportunity for rational drug design against Chagas'' disease. As a first step in the analysis of the parasite enzyme we report its purification and characterization. 1. 2.2 mg trypanothione reductase was extracted from 33 g wet weight of cultured epimastigotes or from 4 g lyophilized cells. The flavoenzyme was purified 2400-fold to homogeneity in three steps with an overall yield of 45%. 2. The enzyme is a dimer with a subunit Mr of 50,000. Using NADPH (Km=5 .mu.M) and trypanothione disulfide (Km=45 .mu.M) as substrates, a turnover number of 14200 min-1 was estimated. Trypanothione reductase, the parasite enzyme, and glutathione reductase, the host enzyme, exhibit mutually exclusive specificities for their respective disulfide substrates. 3. When screening cell cultures or column eulates for the presence of trypanothione reductase, a microassay based on Ellman''s reagent as indicator was used. A mixture of regioisomeric glutathionylspermidine disulfides isolated from Escherichia coli served as substrate in this microassay. 4. Experimentally, the catalytic cycle of the enzyme can be subdivided into the half-reactions Eox + ADPH + H+ .fwdarw. EN2 + NADP+, and EH2 + trypanothione disulfide .fwdarw. Eox + dihydrotrypanothione. This is also true for the crystallized enzyme in the presence of 2 M (NH4)2SO4. 5. The spectral properties of trypanothione reductase both in the oxidized form (Eox) and in the two-electron reduced form (EH2) closely resemble those of human glutathione reductase. Both proteins contain a flavin and a redox-active disulfite at the catalytic site. After reduction of Eox to EH2, trypanthione reductase can be inactivated by specifically alkylating one of the nascent active-site thiols.