KINETICS, REGULATION, AND MECHANISMS OF BIOCHEMICAL REACTIONS

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Reactive oxygen species (ROS) have been implicated as signal transducers that cause an elevation of tyrosine phosphorylated proteins in non-phagocytic cells. This elevation can be achieved by inactivating protein tyrosine phosphatases (PTPs). We and others have shown in vitro that the highly abundant PTP, PTP-1B, is inactivated by ROS via oxidation of its catalytic site C215. We found that superoxide radical anion is a kinetically more efficient and chemically more specific oxidant than hydrogen peroxide for inactivating PTP-1B. The initial oxidized product, the C215 sulfenic derivative, can easily be oxidized further to its irreversible derivative, a reaction that can be prevented by glutathionylation. The S-glutathionylated PTP-1B can then be reactivated by thiotransferase. Thus, a signal transduction mechanism mediated by superoxide radical anion and the participation of glutathione is proposed for the regulation of PTP-1B. This mechanism is supported by the in vivo demonstration that glutathionylated PTP-1B at C215 is formed in A431 cells when they were treated with epidermal growth factor. In addition, the superoxide level is regulated by Mn(II)-superoxide dismutase (MnSOD). We found that a phorbol ester, TPA, induces human MnSOD gene via a protein kinase C-catalyzed phosphorylation of a CREB-1/ATF-1-like factor and not via known redox- sensitive transcription factors, such as NF-kappa-B or AP-1. Hydrogen peroxide has been shown to induce intracellular calcium release through the activation of phospholipase C-gamma-2 (PLC-gamma-2) via both tyrosine phosphorylation of PLC-gamma-2 and activation of phosphatidylinositol 3-kinase. - protein phosphatase, free radical, oxidative stress, fast reaction, vesicles, electroporation, EPR, glycation