Murakami Keiko, Tsubouchi Ryoko, Fukayama Minoru, Yoshino Masataka
Department of Biochemistry, Aichi Medical University School of Medicine, Yazako-Karimata 1-1, Nagakute, Aichi, 480-1195, Japan.
Biometals. 2014 Jun;27(3):551-8. doi: 10.1007/s10534-014-9731-x. Epub 2014 Mar 27.
Effects of copper on the activity and oxidative inactivation of yeast glutathione reductase were analyzed. Glutathione reductase from yeast was inhibited by cupric ion and more potently by cuprous ion. Copper ion inhibited the enzyme noncompetitively with respect to the substrate GSSG and NADPH. The Ki values of the enzyme for Cu(2+) and Cu(+) ion were determined to be 1 and 0.35 μM, respectively. Copper-dependent inactivation of glutathione reductase was also analyzed. Hydrogen peroxide and copper/ascorbate also caused an inactivation with the cleavage of peptide bond of the enzyme. The inactivation/fragmentation of the enzyme was prevented by addition of catalase, suggesting that hydroxyl radical produced through the cuprous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed eight fragments, which were further determined to result from the cleavage of the Met17-Ser18, Asn20-Thr21, Glu251-Gly252, Ser420-Pro421, Pro421-Thr422 bonds of the enzyme by amino-terminal sequencing analysis. Based on the kinetic analysis and no protective effect of the substrates, GSSG and NADPH on the copper-mediated inactivation/fragmentation of the enzyme, copper binds to the sites apart from the substrate-sites, causing the peptide cleavage by hydroxyl radical. Copper-dependent oxidative inactivation/fragmentation of glutathione reductase can explain the prooxidant properties of copper under the in vivo conditions.
分析了铜对酵母谷胱甘肽还原酶活性和氧化失活的影响。酵母中的谷胱甘肽还原酶受到铜离子抑制，亚铜离子的抑制作用更强。铜离子对底物谷胱甘肽二硫化物（GSSG）和烟酰胺腺嘌呤二核苷酸磷酸（NADPH）而言呈非竞争性抑制该酶。该酶对铜离子（Cu(2+)）和亚铜离子（Cu(+)）的抑制常数（Ki）分别测定为1和0.35μM。还分析了铜依赖性的谷胱甘肽还原酶失活情况。过氧化氢以及铜/抗坏血酸盐也会导致该酶失活并伴有肽键断裂。添加过氧化氢酶可防止该酶失活/断裂，这表明通过亚铜离子依赖性的氧还原产生的羟基自由基是导致该酶失活/断裂的原因。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和飞行时间质谱（TOF-MS）分析确认了八个片段，通过氨基末端测序分析进一步确定这些片段是由该酶的甲硫氨酸17-丝氨酸18、天冬酰胺20-苏氨酸21、谷氨酸251-甘氨酸252、丝氨酸420-脯氨酸421、脯氨酸421-苏氨酸422键断裂产生的。基于动力学分析以及底物GSSG和NADPH对铜介导的该酶失活/断裂无保护作用，铜结合在远离底物结合位点的部位，导致肽链被羟基自由基切割。铜依赖性的谷胱甘肽还原酶氧化失活/断裂可以解释体内条件下铜的促氧化特性。
