Sohn Jungsan, Rudolph Johannes
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Biochemistry. 2003 Sep 2;42(34):10060-70. doi: 10.1021/bi0345081.
Cdc25 phosphatases belong to the family of protein tyrosine phosphatases (PTPs) that contain an active-site cysteine and form a phosphocysteine intermediate. Recently, oxidation/reduction of active-site cysteines of PTPs, including Cdc25, has been proposed to serve as a form of reversible regulation for this class of enzymes. Here we provide in vitro evidence that supports the chemical and kinetic competence for oxidation/reduction of the active-site cysteines of Cdc25B and Cdc25C as a mechanism of regulation. Using kinetic measurements and mass spectrometry, we have found that the active-site cysteines of the Cdc25's are highly susceptible to oxidation. The rate of thiolate conversion to the sulfenic acid by hydrogen peroxide for Cdc25B is 15-fold and 400-fold faster than that for the protein tyrosine phosphatase PTP1B and the cellular reductant glutathione, respectively. If not for the presence of an adjacent (back-door) cysteine in proximity to the active-site cysteine in the Cdc25's, the sulfenic acid would rapidly oxidize further to the irreversibly inactivated sulfinic acid, as determined by using kinetic partitioning and mass spectrometry with mutants of these back-door cysteines. Thus, the active-site cysteine is protected by rapid intramolecular disulfide formation with the back-door cysteines in the wild-type enzymes. These intramolecular disulfides can then be rapidly and effectively rereduced by thioredoxin/thioredoxin reductase but not glutathione. Thus, the chemistry and kinetics of the active-site cysteines of the Cdc25's support a physiological role for reversible redox-mediated regulation of the Cdc25's, important regulators of the eukaryotic cell cycle.
Cdc25磷酸酶属于蛋白质酪氨酸磷酸酶（PTP）家族，该家族含有一个活性位点半胱氨酸并形成磷酸半胱氨酸中间体。最近，有人提出包括Cdc25在内的PTP活性位点半胱氨酸的氧化/还原可作为这类酶的一种可逆调节形式。在此，我们提供体外证据，支持将Cdc25B和Cdc25C活性位点半胱氨酸的氧化/还原作为一种调节机制的化学和动力学能力。通过动力学测量和质谱分析，我们发现Cdc25的活性位点半胱氨酸极易被氧化。过氧化氢将Cdc25B的硫醇盐转化为亚磺酸的速率分别比蛋白质酪氨酸磷酸酶PTP1B和细胞还原剂谷胱甘肽快15倍和400倍。通过对这些“后门”半胱氨酸突变体进行动力学分配和质谱分析确定，如果Cdc25中活性位点半胱氨酸附近不存在相邻（“后门”）半胱氨酸，亚磺酸会迅速进一步氧化为不可逆失活的亚磺酸。因此，在野生型酶中，活性位点半胱氨酸通过与“后门”半胱氨酸快速形成分子内二硫键而得到保护。然后，这些分子内二硫键可被硫氧还蛋白/硫氧还蛋白还原酶快速有效地重新还原，但不能被谷胱甘肽还原。因此，Cdc25活性位点半胱氨酸的化学性质和动力学支持了对Cdc25进行可逆氧化还原介导调节的生理作用，Cdc25是真核细胞周期的重要调节因子。
