Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Kiel, Germany.
Chem Biol Interact. 2011 May 30;191(1-3):95-103. doi: 10.1016/j.cbi.2011.01.016. Epub 2011 Jan 21.
Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236-244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K(m) was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k(cat), but a 30-fold higher K(m), and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.
人类羰基还原酶 1 和 3（CBR1 和 CBR3）是短链脱氢/还原酶超家族中的单体 NADPH 依赖性酶。尽管一级结构具有 72%的同源性，但它们在底物特异性上存在显著差异。最近，内源性低分子量 S-亚硝酰化谷胱甘肽 S-亚硝酰基谷胱甘肽（GSNO）已被添加到 CBR1 的广泛底物谱中。本研究最初探讨了 CBR3 是否可以同样还原 GSNO，但事实并非如此。在 CBR1 中有助于谷胱甘肽结合的残基（即 K106Q 和 S97V/D98A）的引入，以及防止 CBR3 中与 C227 形成理论二硫键的 C143S 交换，都不能使 CBR3 产生对 GSNO 的活性。然而，将 CBR3 中的氨基酸 236-244 交换为与 CBR1 对应的氨基酸足以使 CBR3 产生对 GSNO 的催化活性。通过交换 Q142M、C143S、P230W 和 H270S，催化效率进一步提高。因此，先前报道的对羰基化合物还原很重要的相同残基似乎是 CBR1 介导的 GSNO 还原的关键。此外，对于 CBR1 介导的 GSNO 还原，在浓度>5 K(m)时观察到相当大的底物抑制。用 GSNO 处理 CBR1 后去除低分子量化合物会降低 GSNO 还原活性，表明发生了共价修饰。用二硫苏糖醇处理而不是用抗坏血酸处理可以挽救活性，表明 S-谷胱甘肽化而不是 S-亚硝化为潜在机制。由于 C227 先前被鉴定为 CBR1 中的反应性半胱氨酸，因此生成了变体 CBR1 C227S，与野生型蛋白相比，该变体显示出相似的 k(cat)，但 K(m)高 30 倍，且不显示底物抑制。总的来说，这些结果清楚地表明 CBR1 而不是 CBR3 在 GSNO 还原中具有生理作用，从而最终在调节 NO 信号中具有作用。此外，在较高浓度下，GSNO 似乎作为 CBR1 的自杀抑制剂起作用，可能是通过 C227 的谷胱甘肽化。
