KINETICS, REGULATION AND MECHANISMS OF BIOCHEMICAL REACTIONS

EPR光谱和自旋捕获方法被用来鉴定和 监测自由基的形成和利用。 与这些 方法，我们已经表明，铜，锌-超氧化物歧化酶（Cu，Zn-SOD），在 除了其歧化酶活性外，还可以催化 自由基与过氧化氢作为底物。 我们发现， 一些Cu，Zn-SOD突变体，如G93 A和A4 V，与家族性 肌萎缩侧索硬化症（ALS），表现出功能获得性， 它们催化自由基生成的能力，而它们的歧化酶 活动保持不变。 增强的自由基生成 容量是由于过氧化氢Km值的降低 相对于野生型酶的突变体。 核实这一 相关性，我们目前正在过度表达另一种突变体，G37 R， 类似的研究。 此外，有人认为，FALS Cu，Zn-SOD 表现出较高的催化过氧亚硝酸盐介导的酪氨酸活性， 相对于野生型酶的硝化。 然而，我们无法 与人野生型酶相比，证实了这一报告。 一氧化氮与超氧阴离子反应形成过氧亚硝酸根， 氧化应激 我们已经证明过氧亚硝酸盐介导的酪氨酸 蛋白酪氨酸激酶磷酸化位点的硝化不能被 磷酸化。 为了进一步研究酪氨酸硝化的影响， 在其调节功能方面，我们研究了两种src的结合能力， 同源性2（SH 2）识别肽ENAEYLDLDC和CGDNDYIIPL，与 磷脂酶C的N端和C端的SH 2结构域 γ型（PLC-γ）。 使用BIAcore系统，我们 发现磷酸化的ENAEYLDLDC和CGDNDYIIPL结合到 N-末端和C-末端的Kd分别为0.22 μ M和0.33 μ M， 分别 然而，硝化和非磷酸化肽 不能与PLC-γ结合。 这表明，不可逆转的 酪氨酸硝化将永久性地损害 涉及酪氨酸磷酸化的循环级联。 蛋白质酪氨酸磷酸化在信号转导中起着关键作用 转导 除酪氨酸激酶外，据信， 蛋白酪氨酸磷酸酶（PTP），其通过蛋白酪氨酸磷酸酶（PTP）发挥其功能。 在其活性位点半胱氨酸上形成硫代磷酸酯中间体， 参与调节细胞信号， 物种参与其中。 我们正在调查 应激介导的PTP-1 β失活。 利用我们自制的电转化系统，我们正在探索 开发一种快速动力学方法的可能性， 事实上，反应物可以被负载的囊泡分离， 被电场脉冲迅速破坏 然而，人们需要 克服了囊泡制备的异质性问题， 囊泡破裂

EPR spectroscopic and spin-trapping methods were used to identify and monitor the formation and utilization of free radicals. With these methods, we have shown that Cu,Zn-superoxide dismutase (Cu,Zn-SOD), in addition to its dismutase activity, can also catalyze the generation of free radicals with hydrogen peroxide as substrate. We revealed that some Cu,Zn-SOD mutants, e.g. G93A and A4V, associated with familial amyotrophic lateral sclerosis (FALS), exhibit a gain-of-function in their ability to catalyze free radical generation, while their dismutase activity remains unchanged. The enhanced free radical generating capacity is due to the decrease in the Km value for hydrogen peroxide for the mutant relative to the wild type enzyme. To verify this correlation, we are currently overexpressing another mutant, G37R, for a similar study. In addition, it has been suggested that FALS Cu,Zn-SOD exhibits higher activity for catalyzing peroxynitrite-mediated tyrosine nitration relative to the wild type enzyme. However, we are unable to confirm this report when compared with human wild type enzyme. Nitric oxide reacts with superoxide anion to form peroxynitrite under oxidative stress. We have shown that peroxynitrite-mediated tyrosine nitration of the protein tyrosine kinase phosphorylation site cannot be phosphorylated. To further investigate the effect of tyrosine nitration on its regulatory function, we studied the binding capacity of two src homology 2 (SH2) recognition peptides, ENAEYLDLDC and CGDNDYIIPL, to the SH2 domain at the N-terminal and the C-terminal of the phospholipase C type gamma (PLC-gamma), respectively. Using the BIAcore system, we found that the phosphorylated ENAEYLDLDC and CGDNDYIIPL bind to the N-terminal and C-terminal with a Kd of 0.22 microM and 0.33 microM, respectively. However, the nitrated and the unphosphorylated peptides fail to bind to the PLC-gamma. This suggests that the irreversible tyrosine nitration will permanently impair the regulatory function of cyclic cascades involving tyrosine phosphorylation. Protein tyrosine phosphorylation plays a key role in signal transduction. In addition to tyrosine kinase, it is believed that protein tyrosine phosphatase (PTP), which exhibits its function via the formation of a thiophosphate intermediate on its active site cysteine, is participating in mediating cell signaling when reactive oxygen species are involved. We are investigating the oxidative stress-mediated inactivation of PTP-1beta. Using our home-built electroporator system, we are exploring the possibility of developing a rapid kinetic method taking advantage of the fact that reactants can be separated by loaded vesicles, which can be ruptured very rapidly by electric field pulses. However, one needs to overcome the heterogeneity problems due to vesicle preparation and vesicle rupture.