Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is inactivated by S-sulfuration in vitro
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is inactivated by S-sulfuration in vitro
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DOI：
10.1016/j.freeradbiomed.2015.09.007
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发表时间：
2015-12-01
影响因子：
7.4
通讯作者：
Mutus, Bulent
中科院分区：
文献类型：
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作者：
Jarosz, Artur P.;Wei, Wanlei;Mutus, Bulent
硫化氢（H2S）由胱硫醚β-合酶（CBS）和胱硫醚γ-裂解酶（CSE）以及哺乳动物组织中的其他酶酶促产生。这些发现使得H2S成为另一种有毒气体，其充当像NO和CO的气体传递物。H2S被认为通过与蛋白质中的半胱氨酸硫醇反应产生硫化硫醇（-SSH）衍生物来发挥其生物效应。第一个被H2S修饰的蛋白质之一是甘油醛3-磷酸脱氢酶（GAPDH）[1]，其中活性位点半胱氨酸（Cys 152）的S-硫化导致酶活性增加约7倍。在本研究中，我们试图重现这一结果，但没有成功。GAPDH以其还原形式、或过氧化氢形式、或谷胱甘肽二硫化物形式、或亚硝鎓氧化形式与硫化物或多硫化物反应。硫化物对GAPDH活性的降低没有影响，而多硫化物抑制GAPDH至与对照组相似的42%。硫处理后GAPDH在Cys 247处发生S-硫化，多硫处理后在Cys 156和Cys 247处发生S-硫化。没有发现活性位点Cys 152处S-硫化的证据。硫化物和多硫化物都能够恢复谷胱甘肽二硫化物氧化GAPDH的活性，但不能控制未处理的水平。谷胱甘肽二硫化物氧化GAPDH与多硫化物的治疗也产生了半胱氨酸156的S-硫化。用硫化物和多硫化物处理GAPDH的C156 S突变体导致Cys 152的S-硫化，这也引起酶活性的降低而不是增加。计算化学表明，Cys 156的S-硫化可能影响催化Cys 152的位置，使其pKa升高0.5，这可能影响Cys 152的亲核性。目前的研究提出了重大的问题，报告的能力H2S激活GAPDH的硫化其活性位点硫醇，并表明，多硫化物是一个更强的蛋白S-硫化剂比硫化物。(C)2015爱思唯尔公司All rights reserved.
Hydrogen sulfide (H2S) is produced enzymatically by cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE), as well as other enzymes in mammalian tissues. These discoveries have led to the crowning of H2S as yet another toxic gas that serves as a gasotransmitter like NO and CO. H2S is thought to exert its biological effects through its reaction with cysteine thiols in proteins, yielding sulfurated thiol (-SSH) derivatives. One of the first proteins shown to be modified by H2S was glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [1] where the S-sulfuration of the active site cysteine (Cys 152) resulted in similar to 7-fold increase in the activity of the enzyme. In the present study we have attempted to reproduce this result with no success. GAPDH in its reduced, or hydrogen peroxide, or glutathione disulfide, or nitrosonium oxidized forms was reacted with sulfide or polysulfides. Sulfide had no effect on reduced GAPDH activity, while polysulfides inhibited GAPDH to similar to 42% of control. S-sulfuration of GAPDH occurred at Cys 247 after sulfide treatment, Cys 156 and Cys 247 after polysulfide treatment. No evidence of S-sulfuration at active site Cys 152 was discovered. Both sulfide and polysulfide was able to restore the activity of glutathione disulfide oxidized GAPDH, but not to control untreated levels. Treatment of glutathione disulfide oxidized GAPDH with polysulfide also produced S-sulfuration of Cys 156. Treatment of a C156S mutant of GAPDH with sulfide and polysulfide resulted in S-sulfuration of Cys 152, which also caused a decrease and not an increase in enzymatic activity. Computational chemistry shows S-sulfuration of Cys 156 may affect the position of catalytic Cys 152, raising its pKa by 0.5, which may affect the nucleophilicity of Cys 152. The current study raises significant questions about the reported ability of H2S to activate GAPDH by the sulfuration of its active site thiol, and indicates that polysulfide is a stronger protein S-sulfurating agent than sulfide. (C) 2015 Elsevier Inc. All rights reserved.