Jockers-Scherübl M C, Schirmer R H, Krauth-Siegel R L
Institut für Biochemie II der Universität Heidelberg.
Eur J Biochem. 1989 Mar 15;180(2):267-72. doi: 10.1111/j.1432-1033.1989.tb14643.x.
Trypanothione reductase of Trypanosoma cruzi is a key enzyme in the antioxidant metabolism of the parasite. Here we report on the enzymic and pharmacological properties of trypanothione reductase using glutathionylspermidine disulfide as a substrate. 1. Both pH optimum (7.5) and the ionic strength optimum (at 30 mM) are unusually narrow for this enzyme. 40 mM Hepes, 1 mM EDTA, pH 7.5 was chosen as a standard assay buffer because in this system the kcat/Km ratio had the highest values for both natural substrates, glutathionylspermidine disulfide (2.65 x 10(6) M-1 s-1) and trypanothione disulfide (4.63 x 10(6) M-1 s-1). 2. Using the standardized assay, trypanothione reductase and the phylogenetically related host enzyme, human glutathione reductase, were studied as targets of inhibitors. Both enzymes, in their NADPH-reduced forms, were irreversibly modified by the cytostatic agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Nifurtimox, the drug used in the treatment of Chagas' disease, is a stronger inhibitor of glutathione reductase (Ki = 40 microM) than of trypanothione reductase (IC50 = 200 microM). 3. Of the newly synthesized trypanocidal compounds [Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) Proc. Natl Acad. Sci., 85, 5374-5378] a nitrofuran derivative, 2-(5-nitro-2-furanylmethylidene)-N,N'-[1,4-piperazinediylbis (1,3-propanediyl)]bishydrazinecarboximidamide tetrahydrobromide, was found to be a better inhibitor for trypanothione reductase (Ki = 0.5 microM) than for glutathione reductase (IC50 = 10 microM). A naphthoquinone derivative, 2,3-bis[3-(2-amidinohydrazono)-butyl]-1,4-naphthoquinone dihydrochloride, turned out to be both an inhibitor (IC50 = 1 microM) and an NADPH-oxidation-inducing substrate (Km = 14 microM). This effect was not observed with human glutathione reductase. Such compounds which lead to oxidative stress by more than one mechanism in the parasite are promising starting points for drug design based on the three-dimensional structures of glutathione and trypanothione reductases.
克氏锥虫的锥虫硫醇还原酶是该寄生虫抗氧化代谢中的关键酶。在此，我们报道了以谷胱甘肽亚精胺二硫化物为底物时锥虫硫醇还原酶的酶学和药理学特性。1. 该酶的最适pH（7.5）和最适离子强度（30 mM）范围异常狭窄。选择40 mM Hepes、1 mM EDTA、pH 7.5作为标准测定缓冲液，因为在该体系中，天然底物谷胱甘肽亚精胺二硫化物（2.65×10⁶ M⁻¹ s⁻¹）和锥虫硫醇二硫化物（4.63×10⁶ M⁻¹ s⁻¹）的kcat/Km比值均最高。2. 使用标准化测定方法，研究了锥虫硫醇还原酶和系统发育相关的宿主酶——人谷胱甘肽还原酶作为抑制剂的作用靶点。两种酶在其NADPH还原形式下，均被细胞抑制剂1,3 - 双（2 - 氯乙基）- 1 - 亚硝基脲（BCNU）不可逆修饰。用于治疗恰加斯病的药物硝呋莫司，对谷胱甘肽还原酶（Ki = 40 μM）的抑制作用比对锥虫硫醇还原酶（IC50 = 200 μM）更强。3. 在新合成的杀锥虫化合物中[亨德森，G. B.，乌尔里希，P.，费尔兰姆，A. H.，罗森伯格，I.，佩雷拉，M.，塞拉，M. & 塞拉米，A.（1988年）《美国国家科学院院刊》，85，5374 - 5378]，发现一种硝基呋喃衍生物，2 -（5 - 硝基 - 2 - 呋喃基亚甲基）- N，N'-[1,4 - 哌嗪二基双（1,3 - 丙二基）]双肼甲酰胺四氢溴化物，对锥虫硫醇还原酶（Ki = 0.5 μM）的抑制作用比对谷胱甘肽还原酶（IC50 = 10 μM）更强。一种萘醌衍生物，2,3 - 双[3 -（2 - 脒基肼基）- 丁基]- 1,4 - 萘醌二盐酸盐，结果显示既是一种抑制剂（IC50 = 1 μM），又是一种诱导NADPH氧化的底物（Km = 14 μM）。人谷胱甘肽还原酶未观察到这种效应。这类能通过多种机制在寄生虫中导致氧化应激的化合物，是基于谷胱甘肽和锥虫硫醇还原酶三维结构进行药物设计的有前景的起点。
