Del Boccio G, Pennelli A, Whitehead E P, Lo Bello M, Petruzzelli R, Federici G, Ricci G
Institute of Biochemical Sciences, University of Chieti G. D'Annunzio, Italy.
J Biol Chem. 1991 Jul 25;266(21):13777-82.
7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with two thiol groups of the dimeric horse erythrocyte glutathione transferase at pH 5.0, with strong inactivation reversible on dithiothreitol treatment. The inactivation kinetic follows a biphasic pattern, similar to that caused by other thiol reagents as recently reported. Both S-methylglutathione and 1-chloro-2,4-dinitrobenzene protect the enzyme from inactivation. Analysis of the reactive SH group-containing peptide gives the sequence Ala-Ser-Cys-Leu-Tyr, identical with that of the peptide that contains the reactive cysteine 47 of the human placental transferase. In the presence of glutathione, the enzyme is not inactivated by this reagent, but it catalyzes its conjugation to glutathione. At higher pH values, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacts with 2 tyrosines/dimer and lysines, as well as with cysteines. Reaction with lysine seems essentially without effect on activity; whether the reactive tyrosines are important for activity could not be determined using this reagent only. However, 2 tyrosines among the 4 that are nitrated by tetranitro-methane are important for activity.
7-氯-4-硝基苯并-2-恶唑-1,3-二氮唑在pH 5.0时与二聚体马红细胞谷胱甘肽转移酶的两个巯基反应，导致强烈失活，用二硫苏糖醇处理后可逆转。失活动力学呈双相模式，与最近报道的其他巯基试剂引起的情况相似。S-甲基谷胱甘肽和1-氯-2,4-二硝基苯都能保护该酶不被失活。对含反应性巯基的肽段进行分析，得到的序列为Ala-Ser-Cys-Leu-Tyr，与含有人胎盘转移酶反应性半胱氨酸47的肽段序列相同。在谷胱甘肽存在的情况下，该酶不会被这种试剂失活，但它会催化其与谷胱甘肽结合。在较高的pH值下，7-氯-4-硝基苯并-2-恶唑-1,3-二氮唑与每个二聚体中的2个酪氨酸、赖氨酸以及半胱氨酸反应。与赖氨酸的反应似乎对活性基本没有影响；仅使用这种试剂无法确定反应性酪氨酸对活性是否重要。然而，被四硝基甲烷硝化的4个酪氨酸中有2个对活性很重要。
