Oudot C, Jault J M, Jaquinod M, Negre D, Prost J F, Cozzone A J, Cortay J C
Institut de Biologie et Chimie des Protéines, CNRS, Lyon, France.
Eur J Biochem. 1998 Dec 1;258(2):579-85. doi: 10.1046/j.1432-1327.1998.2580579.x.
The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA-mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.
大肠杆菌异柠檬酸脱氢酶激酶/磷酸酶与ATP类似物5'-[对-(氟磺酰基)苯甲酰基]腺苷（FSBA）预先孵育后，其ATP酶活性迅速丧失。5 mM ATP或5 mM ADP加Mg2+的存在可防止这种失活，而随后加入二硫苏糖醇可使其完全逆转，从而表明半胱氨酸残基参与了这一过程。在失活过程中，每摩尔异柠檬酸脱氢酶激酶/磷酸酶结合约2摩尔[3H]FSBA。然而，预先与ATP孵育或随后加入二硫苏糖醇均未显著改变这种结合。这表明FSBA介导的异柠檬酸脱氢酶激酶/磷酸酶失活是通过二硫键的形成发生的。因此，质谱分析显示，加入FSBA后，半胱氨酸356和半胱氨酸523之间形成了二硫键。半胱氨酸356突变为丝氨酸使该酶对FSBA处理不敏感，表明半胱氨酸356是该类似物的主要作用靶点。然而，半胱氨酸523突变为丝氨酸的突变体仍可被FSBA失活，质谱分析表明这是由于半胱氨酸356和半胱氨酸480之间形成了新的二硫键。
