Área de Química Física, Universidad de Almería, Crta. de Sacramento s/n, 04120 Almería, Spain.
Chembiochem. 2012 Jul 23;13(11):1594-604. doi: 10.1002/cbic.201200210. Epub 2012 Jun 27.
Human glutathione S-transferase P1-1 (hGST P1-1) is involved in cell detoxification processes through the conjugation of its natural substrate, reduced glutathione (GSH), with xenobiotics. GSTs are known to be overexpressed in tumors, and naturally occurring isothiocyanates, such as benzyl isothiocyanate (BITC), are effective cancer chemopreventive compounds. To identify and characterize the potential inhibitory mechanisms of GST P1-1 induced by isothiocyanate conjugates, we studied the binding of GST P1-1 and some cysteine mutants to the BITC-SG conjugate as well as to the synthetic S-(N-benzylcarbamoylmethyl)glutathione conjugate (BC-SG). We report here the inactivation of GST P1-1 through the covalent modification of two Cys47 residues per dimer and one Cys101. The evidence has been compiled by isothermal titration calorimetry (ITC) and electrospray ionization mass spectrometry (ESI-MS). ITC experiments suggest that the BITC-SG conjugate generates adducts with Cys47 and Cys101 at physiological temperatures through a corresponding kinetic process, in which the BITC moiety is covalently bound to these enzyme cysteines through an S-thiocarbamoylation reaction. ESI-MS analysis of the BITC-SG incubated enzymes indicates that although the Cys47 in each subunit is covalently attached to the BITC ligand moiety, only one of the Cys101 residues in the dimer is so attached. A plausible mechanism is given for the emergence of inactivation through the kinetic processes with both cysteines. Likewise, our molecular docking simulations suggest that steric hindrance is the reason why only one Cys101 per dimer is covalently modified by BITC-SG. No covalent inactivation of GST P1-1 with the BC-SG inhibitor has been observed. The affinities and inhibitory potencies for both conjugates are high and very similar, but slightly lower for BC-SG. Thus, we conclude that the presence of the sulfur atom from the isothiocyanate moiety in BITC-SG is crucial for its irreversible inhibition of GST P1-1.
人谷胱甘肽 S-转移酶 P1-1（hGST P1-1）通过将其天然底物还原型谷胱甘肽（GSH）与外源性化学物质缀合，参与细胞解毒过程。 GST 在肿瘤中表达过度，而天然存在的异硫氰酸酯，如苄基异硫氰酸酯（BITC），是有效的癌症化学预防化合物。为了鉴定和表征异硫氰酸酯缀合物诱导的 GST P1-1 的潜在抑制机制，我们研究了 GST P1-1 与一些半胱氨酸突变体与 BITC-SG 缀合物以及合成的 S-（N-苄基氨基甲酰基甲基）谷胱甘肽缀合物（BC-SG）的结合。我们在这里报告 GST P1-1 通过二聚体每个二聚体的两个 Cys47 残基和一个 Cys101 的共价修饰而失活。通过等温滴定量热法（ITC）和电喷雾电离质谱（ESI-MS）收集了证据。 ITC 实验表明，BITC-SG 缀合物在生理温度下通过相应的动力学过程与 Cys47 和 Cys101 生成加合物，其中 BITC 部分通过 S-硫代氨甲酰化反应与这些酶半胱氨酸共价结合。 BITC-SG 孵育酶的 ESI-MS 分析表明，尽管每个亚基的 Cys47 都与 BITC 配体部分共价连接，但二聚体中的仅一个 Cys101 残基与之连接。给出了通过两个半胱氨酸的动力学过程出现失活的合理机制。同样，我们的分子对接模拟表明，空间位阻是 BITC-SG 仅使每个二聚体的一个 Cys101 残基发生共价修饰的原因。没有观察到 GST P1-1 与 BC-SG 抑制剂的共价失活。两种缀合物的亲和力和抑制效力均很高且非常相似，但 BC-SG 略低。因此，我们得出结论，BITC-SG 中异硫氰酸酯部分的硫原子对于其对 GST P1-1 的不可逆抑制至关重要。
