Araki Shoma, Takata Tsuyoshi, Yoon Sunghyeon, Kasamatsu Shingo, Ihara Hideshi, Nakagawa Hidehiko, Akaike Takaaki, Tsuchiya Yukihiro, Watanabe Yasuo
Department of Pharmacology, Showa Pharmaceutical University, Machida 194-8543, Japan.
Department of Redox Molecular Medicine, Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan.
Antioxidants (Basel). 2025 Sep 13;14(9):1113. doi: 10.3390/antiox14091113.
Cystathionine γ-lyase (CSE) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the final step of the transsulfuration pathway, converting cystathionine into cysteine. Additionally, CSE is also essential for the formation of cysteine hydropolysulfide (Cys-S-(S)n-H), known as supersulfides, by metabolizing cystine under pathological conditions. We previously reported that, during cystine metabolism, CSE undergoes self-inactivation through polysulfidation at the Cys136 residue. Here, contrary to the anticipated role of L-S-nitrosocysteine (L-CysNO) as a nitric oxide (NO) donor, we demonstrate that it serves as a substrate for CSE and that its metabolites inhibit the activity of the enzyme during L-CysNO metabolism. The in vitro incubation of CSE-but not the Cys136/171Val mutant-with L-CysNO resulted in the dose-dependent inhibition of supersulfide production, which was not reversed by the reducing agents. Notably, CSE activity remained unchanged upon preincubation with other NO donors, such as S-nitrosoglutathione or D-CysNO, but was inhibited when coincubated with cysteine. Furthermore, when PLP was removed from the CSE/L-CysNO premix, L-CysNO no longer inhibited CSE activity, suggesting that CSE metabolizes L-CysNO and that its metabolites contribute to enzyme inactivation. Indeed, we identified thionitrous acid and pyruvate as the primary CSE/L-CysNO reaction products. Thus, we establish L-CysNO as a CSE substrate and demonstrate that its metabolites act as enzyme inhibitors through a novel irreversible modification at the Cys136/171 residues.
胱硫醚γ-裂解酶（CSE）是一种依赖于磷酸吡哆醛（PLP）的酶，催化转硫途径的最后一步，将胱硫醚转化为半胱氨酸。此外，在病理条件下，CSE对于通过代谢胱氨酸形成半胱氨酸氢多硫化物（Cys-S-(S)n-H，即超硫化物）也至关重要。我们之前报道过，在胱氨酸代谢过程中，CSE通过Cys136残基的多硫化作用发生自失活。在此，与L-亚硝基半胱氨酸（L-CysNO）作为一氧化氮（NO）供体的预期作用相反，我们证明它是CSE的底物，并且其代谢产物在L-CysNO代谢过程中抑制该酶的活性。CSE（而非Cys136/171Val突变体）与L-CysNO的体外孵育导致超硫化物生成的剂量依赖性抑制，还原剂不能逆转这种抑制。值得注意的是，与其他NO供体（如亚硝基谷胱甘肽或D-CysNO）预孵育时，CSE活性保持不变，但与半胱氨酸共孵育时则受到抑制。此外，当从CSE/L-CysNO预混物中去除PLP时，L-CysNO不再抑制CSE活性，这表明CSE代谢L-CysNO，并且其代谢产物导致酶失活。实际上，我们确定硫亚硝酸和丙酮酸是CSE/L-CysNO反应的主要产物。因此，我们确定L-CysNO为CSE的底物，并证明其代谢产物通过在Cys136/171残基处进行新的不可逆修饰而作为酶抑制剂。
