Takakura K, Beckman J S, MacMillan-Crow L A, Crow J P
Department of Anesthesiology and Reanimatology, Fukui Medical University, Fukui, Japan.
Arch Biochem Biophys. 1999 Sep 15;369(2):197-207. doi: 10.1006/abbi.1999.1374.
Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of </=0.9 microM. PTP inactivation was also seen with equivalent concentrations of peroxynitrite generated by SIN-1, indicating that bolus peroxynitrite and cogeneration of superoxide and nitric oxide were equipotent. Rate constants for peroxynitrite-mediated PTP inactivation were determined by competition with cysteine and were among the fastest rates yet seen for reaction of peroxynitrite with any biological molecules. The bimolecular reaction rates for CD45, LAR, and PTP1B were 2.0 x 10(8), 2.3 x 10(7), and 2.2 x 10(7) M(-1) s(-1), respectively. Inactivation by peroxynitrite was essentially irreversible as incubation with dithiothreitol (DTT) restored less than 10% of the original phosphatase activity. Prolonged treatment with 0.4 mM DETA NONOate, which generated a steady-state concentration of 2 microM nitric oxide, was only slightly inhibitory. S-Nitrosoglutathione (1.0 mM) inhibited PTPs by approximately 50% after 30 min and the inhibition was completely reversed by DTT. Nitrotyrosine immunoblots of peroxynitrite-treated PTP1B revealed that peroxynitrite completely inactivated PTP1B prior to the appearance of protein tyrosine nitration. Peroxynitrite anion is structurally similar to phosphate anion both in terms of molecular diameter and charge. Thus, the extreme vulnerability of these PTPs to peroxynitrite-mediated inactivation is consistent with attraction of peroxynitrite anion to the active site and subsequent oxidation of the essential thiolate. These findings suggest that any PTP possessing the CXXXXXR active-site sequence could potentially be inactivated by peroxynitrite in vivo resulting in a net increase in tyrosine phosphorylation and profound effects on phosphotyrosine-dependent signaling cascades.
蛋白酪氨酸磷酸酶（PTPs）在活性位点含有一个必需的硫醇，它可能易受一氧化氮衍生的生物氧化剂的攻击。我们在体外评估了过氧亚硝酸盐、一氧化氮和S-亚硝基谷胱甘肽对三种人酪氨酸磷酸酶活性的影响。受体样T细胞酪氨酸磷酸酶（CD45）、非受体样酪氨酸磷酸酶PTP1B和白细胞抗原相关（LAR）磷酸酶在不到1秒的时间内均被过氧亚硝酸盐不可逆地失活，IC(50)值≤0.9微摩尔。由SIN-1产生的等效浓度的过氧亚硝酸盐也能使PTP失活，这表明一次性加入的过氧亚硝酸盐以及超氧化物和一氧化氮的同时产生具有同等效力。过氧亚硝酸盐介导的PTP失活的速率常数通过与半胱氨酸竞争来确定，是过氧亚硝酸盐与任何生物分子反应中迄今观察到的最快速率之一。CD45、LAR和PTP1B的双分子反应速率分别为2.0×10(8)、2.3×10(7)和2.2×10(7) M(-1) s(-1)。过氧亚硝酸盐引起的失活基本上是不可逆的，因为用二硫苏糖醇（DTT）孵育后恢复的原始磷酸酶活性不到10%。用0.4 mM二乙三胺 NONOate进行长时间处理，其产生的一氧化氮稳态浓度为2微摩尔，仅有轻微抑制作用。S-亚硝基谷胱甘肽（1.0 mM）在30分钟后使PTPs抑制约50%，且这种抑制作用可被DTT完全逆转。对过氧亚硝酸盐处理的PTP1B进行硝基酪氨酸免疫印迹分析显示，在蛋白质酪氨酸硝化出现之前，过氧亚硝酸盐已使PTP1B完全失活。过氧亚硝酸根阴离子在分子直径和电荷方面与磷酸根阴离子在结构上相似。因此，这些PTPs对过氧亚硝酸盐介导的失活的极端敏感性与过氧亚硝酸根阴离子被吸引到活性位点以及随后必需硫醇盐的氧化是一致的。这些发现表明，任何具有CXXXXXR活性位点序列的PTP在体内都可能被过氧亚硝酸盐失活，从而导致酪氨酸磷酸化净增加，并对磷酸酪氨酸依赖性信号级联产生深远影响。
